Animal Reproduction Science 164 (2016) 47–56
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Effects of pH during liquid storage of goat semen on sperm viability and fertilizing potential Chang-He Liu 1 , Hai-Bo Dong 1 , Dong-Li Ma 1 , You-Wei Li, Dong Han, Ming-Jiu Luo, Zhong-Le Chang ∗ , Jing-He Tan ∗ College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an City 271018, P.R. China
a r t i c l e
i n f o
Article history: Received 29 August 2015 Received in revised form 16 October 2015 Accepted 6 November 2015 Available online 10 November 2015 Keywords: Liquid semen storage Extender pH Sperm motility Fertilizing potential Goat
a b s t r a c t A specific problem in goat semen preservation is the detrimental effect of seminal plasma on sperm viability in extenders containing yolk or milk. Thus, the use of chemically defined extenders will have obvious advantages. Although previous studies indicate that the initial pH of an extender is crucial to sustain high sperm motility, changes in extender pH during long-term semen storage have not been observed. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus important for protocol optimization for long-term liquid semen preservation. The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually, and a strong correlation was observed between the two. Whereas increasing the initial extender pH from 6.04 to 6.25 or storage with stabilized pH improved, storage with artificially lowered pH impaired sperm motility. Extender renewal improved sperm motility by maintaining a stable pH. Sperm coating with chicken (Gallus gallus) egg yolk improved motility by increasing tolerance to pH decline. A new extender (n-mZAP) with a higher buffering capacity was formulated, and n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than the currently used mZAP extender did. Goat semen liquid-stored for 12 d in n-mZAP produced pregnancy and kidding rates similar to those obtained with freshly collected semen following artificial insemination. In conclusion, maintenance of a stable pH during liquid semen storage dramatically improved sperm viability and fertilizing potential. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Liquid-stored semen can be an alternative to frozenthawed semen for artificial insemination (AI), because semen cryopreservation is an expensive process. However, although much research has already been conducted
∗ Corresponding authors. E-mail addresses: [email protected] (Z.-L. Chang), [email protected] (J.-H. Tan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.anireprosci.2015.11.011 0378-4320/© 2015 Elsevier B.V. All rights reserved.
to prolong the in vitro viability and fertilizing potential of stored liquid semen, limited improvements have been achieved in different species (Weitze, 1990; De Pauw et al., 2003; Leboeuf et al., 2003). A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of the spermatozoa in extenders containing egg yolk or milk (Leboeuf et al., 2000). The use of milk- or yolk-containing extenders in this species therefore requires the removal of the seminal plasma by washing before semen dilution. Because washing is a complex and time-consuming process, and it causes damage (Harrison and White, 1972) and some loss (Corteel,
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1981) of spermatozoa, the use of chemically defined extenders will have obvious advantages in liquid storage of goat semen. However, reports on the liquid storage of goat semen in chemically defined extenders which contain neither milk nor yolk are few (Leboeuf et al., 2003; Paulenz et al., 2005); so are reports on cryopreservation of goat spermatozoa in such chemically defined media (Kundu et al., 2000, 2002; Khalifa and El-Saidy, 2006). One study (Xu et al., 2009) has shown that the self-made mZAP extender, formulated based on the Zorlesco extender (Gottardi et al., 1980) and the Androhep® extender (Weitze, 1990) used for liquid storage of porcine semen, performed the best among the extenders tested. A forward sperm motility of over 30% was maintained for 9 d and successful pregnancies were induced after AI with semen stored for 7 d when goat semen was diluted and stored in the mZAP extender. Another study (Zhao et al., 2009) examined effects of semen cooling velocity, sperm coating and extender renewal on sperm viability during goat semen storage. With the optimized protocol, a sperm motility of 48% was maintained for 13 d, and an in vitro-fertilizing potential similar to that of fresh semen was maintained for 11 d. The pH of freshly ejaculated boar semen is between 7.2 and 7.5 (Johnson et al., 2000). When glucose was present in the extender, all visible compartments of the boar spermatozoa as well as the extender were acidified to pH 6.2 within 20 h (Kamp et al., 2003). Although it is recognized that a lower pH can reduce sperm motility and metabolic activity, which is good for keeping spermatozoa viable during manipulation or preservation (Johnson et al., 2000), metabolic acidosis may prevent prolonged storage if no proper buffer is present in the extenders. Thus, our previous study demonstrated that whereas 6.04 was the most suitable pH for liquid storage of goat semen, sperm viability decreased significantly when semen was stored at either pH 6.61 or pH 5.54 (Xu et al., 2009). While these results and those from other studies (Maxwell and Salamon, 1993; Johnston et al., 2000) indicate that the initial pH of an extender is crucial to sustain high sperm motility during semen manipulation or storage, changes in semen pH during longterm storage have not been reported. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus of great importance for protocol optimization for long-term liquid semen preservation. The objective of this study was to observe the effect of pH during liquid semen storage in chemically defined extenders on goat sperm viability and fertilizing potential. Experiment 1 monitored and correlated semen pH and sperm motility changes during the storage of coated or non-coated spermatozoa with or without extender renewal. In experiment 2, effects of artificially adjusting semen pH on sperm motility were observed. The adjustments made included stabilizing or lowering pH during storage and increasing the initial extender pH. In experiment 3, a new extender that possesses a higher buffering capacity was developed and its effect on sperm function and fertilizing potential (AI outcomes) was evaluated.
2. Materials and methods 2.1. Ethics statement The experimental procedures were approved by the Animal Care and Use Committee of the Shandong Agricultural University P. R. China (Permit number: SDAUA-2001-001). All chemicals and media used were purchased from Sigma Chemical Co (St. Louis, MO, USA), unless specified otherwise. 2.2. Animals and semen collection The study was conducted at the Animal Station of the Shandong Agricultural University, Shandong Province (122◦ to 114 ◦ E; 34◦ to 38 ◦ N) of China. Male goats were kept in sheltered pens separated from females, and fed hay and concentrate, with water available ad libitum. The Lubei White male goats (n = 5) used in this study were of meat breed. Male goats aged between 2 and 4 yr were trained to ejaculate into an artificial vagina at a doe mount. Semen collection was scheduled to be at 3-d intervals. The ejaculate was collected into a pre-warmed empty tube. 2.3. Extenders The mZAP extender, which contained 63.9 mM glucose, 80.6 mM fructose, 39.8 mM sodium citrate, 5.1 mM ethylenediaminetetraacetic acid (EDTA), 14.9 mM NaHCO3 , 37.8 mM Hepes, 0.25 g/100 mL polyvinyl alcohol (PVA), 5000 IU/100 mL penicillin and 0.1 g/100 mL streptomycin, was prepared as reported previously (Xu et al., 2009). The new mZAP (n-mZAP) extender contained the same constituents as the mZAP did except that NaHCO3 and Hepes in the mZAP extender was replaced with 2-morpholinoethanesulfonic acid (MES) as buffers. The pH of the extenders was adjusted to 6.04 with 1N HCl unless specified otherwise. 2.4. Sperm coating and semen dilution and packaging In experiments not involving sperm coating, ejaculates of original volume were 1:10 diluted with pre-warmed (35 ◦ C) extender. In the sperm-coating experiment, ejaculates were 1:1 diluted with the extender supplemented with 20% chicken (Gallus gallus) egg yolk. Within 5 min, the coated ejaculates were centrifuged at 30 ◦ C for 10 min at 200 × g. Following removal of the supernatant, the coated ejaculates were 1:10 diluted with pre-warmed extender. Both the non-coated and coated ejaculates that had been 1:10 diluted were packaged in 1.5-mL microfuge tubes (0.5 mL semen per tube) and the tubes were then placed in a water bath immediately after packaging and maintained at 35 ◦ C before cooling and storage. 2.5. Semen cooling and storage After packaging, the semen was cooled to and stored at 5 ◦ C in an incubator. Cooling down to 5 ◦ C was achieved by incubating at 5 ◦ C the storage tubes with extended semen
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in a 200-mL pre-warmed (30 ◦ C) water bath as described in our previous study (Zhao et al., 2009). 2.6. Semen pH measurement and sperm quality assessment After semen samples for sperm quality assessment were taken out, the rest semen in the storage tube was centrifuged at 4 ◦ C for 10 min at 500 × g. At the end of the centrifugation, supernatant was collected for pH measurement. The pH was measured using a Denver Instrument pH Meter (UB-7, Denver Instrument, 5 Orville Dr. Bohemia, NY 11716) equipped with a 15-L probe. Sperm motility was analyzed by a computer-assisted sperm analyzer (CASA) system (Sperm Class Analyzer, Microptic SL, Barcelona, Spain). For analysis, 100 L semen samples were mixed with 200 L pre-warmed (37 ◦ C) mZAP extender. Then, 5 L of the sperm suspension (about 60–120 million cells/mL) was placed on a pre-warmed microscope slide, and overlaid with a 22 mm2 coverslip. The slide was maintained at 37 ◦ C during analysis by a heated slide warmer (TOKAT HIT MATS-U55R30, TOKAI HIT CO., LTD, Japan) and observed with the Nikon phase contrast microscope (Eclipse E-200, Nikon Co., Tokyo, Japan) at 200 × magnification. Several fields of view were captured and at least 1000 spermatozoa were counted in each analysis. With respect to the setting parameters, the system has a specific setup for goat sperm evaluation. It was set up as follows: VCL (m/s): 10 < slow < 40 < medium < 75 < rapid; VAP (m/s): 10 < slow < 40 < medium < 75 < rapid; LIN: circular < 50%; Progressivity: 80% of STR. The system issued a sperm motility classification according to the WHO standard parameters as follows. Type A, Rapid progressive; Type B, Slow progressive; Type C, Non- progressive; and Type D, Immotile. The four categories are expressed in percentages. Percentage of motile spermatozoa: A+B+C; Percentage of progressive motile spermatozoa: A+B. In this study, the term “motility” was used to refer percentage of progressive motile spermatozoa. Membrane integrity of spermatozoa was evaluated by the osmotic resistance test (ORT) (Revell and Mrodeb, 1994; Zhou et al., 2004). Briefly, 25 L of semen was added to 200 L of pre-warmed hypo-osmotic solution and mixed thoroughly. After incubation for 45 min at 38.5 ◦ C, 300 L of 2% glutaraldehyde was added. Then, 10 L of the sperm mixture was placed on a hemocytometer and examined under a phase-contrast microscope at a magnification of 400×. A positive response to the test was evident by the coiling of the sperm tail. Both the total number of spermatozoa and the number of spermatozoa with coiled tails in each medium square was counted. The counting was carried out on individual spermatozoa in five to six different medium squares until 200 cells had been counted. Acrosomal intactness was assessed by Coomassie Blue G-250 staining (Larson and Miller, 1999). Briefly, 50 L of semen was added to 1 mL of saline, mixed and centrifuged at 200 × g for 15 s. The pellet was resuspended in 1 mL of paraformaldehyde in PBS and fixed for 30 min. The spermatozoa were washed by centrifugation, and the pellet was resuspended in PBS and spread on a slide for air-drying. The smear was stained for 5 min with 0.22% Coomassie
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Blue G-250 and washed in water. After air-drying, the smear was observed for sperm acrosomal status. The intact acrosomes were stained intensely blue in color, but the spermatozoa that had lost acrosome integrity lacked Coomassie staining over the acrosomal region. The number of spermatozoa with intact acrosomes and the total number of spermatozoa were counted in different fields. 2.7. Artificial insemination Cycling Lubei White does, aged 2–3 yr, were used. Estrus was checked with a vasectomized goats twice a day at 06:00 and 18:00 h. Females in heat were cervically inseminated with stored semen. The inseminating dose was 0.3 mL semen containing 8.5 × 108 progressively motile spermatozoa. Does that began estrus in the morning were first inseminated in the evening the same day, while those found in heat in the evening were inseminated for the first time in the following morning. The does were inseminated for the second time at 12 h after the first insemination. 2.8. Pregnancy determination via ultrasonography Ultrasonography was performed as reported previously (Martínez et al., 1998). Does were restrained in the standing position in a sqeeze chute. After fecal pellets were removed, conventional ultrasound gel (about 50 mL) was inserted with a syringe in to the rectum to act as a coupling medium between the rectal wall and transducer. The transducer was inserted and manipulated in the rectum by external control of the extension. Each dose was scanned transrectally by using a real-time ultrasound scanner equipped with a 5–7.5–9 MHz alternative linear array transducer (HS-2000, Tokyo, Japan) at 25 d post insemination. Once the embryonic vesicle and heart beat were detected, the doe was considered pregnant. If only the embryonic vesicle was detected, the doe was examined again 10 d later to detect fetus; a doe was considered pregnant only when both the vesicle and fetus were detected. Pregnant does were kept until parturition. 2.9. Experimental design Each treatment was repeated 3 times using ejaculates from three different male goats, and thus, each replicate contained one ejaculate from each male goat. Ejaculates from different goats were processed and stored individually. For example, an ejaculate from Goat A, which had a volume of 1 mL, was packaged and stored in 22 storage tubes each containing 0.5 mL semen. Sperm quality and semen pH on day 0 of storage were examined when the packaged semen was cooled to 5 ◦ C, and they were examined every 24 or 48 h of storage thereafter. Every time, one tube was taken from each treatment for examination of pH and sperm quality. Experiment 1. Monitoring changes in semen pH and sperm motility during liquid semen storage under different conditions 1. Changes in sperm motility and semen pH during liquid storage of coated or non-coated semen. Non-coated and
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coated semen samples were stored in the mZAP extender. Sperm motility and semen pH were examined every 24 h of the storage. 2. Effects of extender renewal on semen pH and sperm motility during liquid semen storage. Coated semen was stored in the mZAP extender, and half of the extender was renewed every 48 h during semen storage. To renew the extender, 0.25 mL of supernatant was removed with a pipette from each storage tube and the same amount of fresh extender that had been pre-cooled to 5 ◦ C was injected into the same tube. The procedure was performed with caution, so as not to disturb the sperm pellet. Sperm motility and extender pH were measured immediately before each extender renewal. Experiment 2. Evaluating the effect of artificial pH adjustments on sperm motility during liquid semen storage. 1. Effects of pH stabilization on sperm motility during liquid semen storage. Non-coated and coated semen were stored in the mZAP extender. Whereas semen in the pH non-stabilizing group was stored undisturbed without any manipulation, the pH of semen in the pH stabilizing group was adjusted to 6.04 everyday during the storage. To stabilize semen pH, one storage tube was taken from each treatment every 24 h of storage. Then, 0.25 mL supernatant was collected from the tube. After pH measurement, the pH of the collected supernatant was adjusted by adding increasing amounts of 1N NaOH. The amount of NaOH used was recorded when the pH reached 6.04 and used for pH adjustment in the remaining tubes of the same treatment on the same day. 2. Effects of artificially lowered pH on sperm motility during liquid semen storage. To store semen with artificially lowered extender pH during storage, an extender renewal system was adopted with half of the extender renewed every 48 h during the storage. While the extender was replaced with fresh regular mZAP (pH 6.04) in the pH non-lowered group, it was renewed using mZAP with pre-adjusted pH values in the pH-lowered group. For non-coated semen, the pH values of mZAP extender used for renewal were adjusted to 6.01, 5.91, 5.82, 5.77, 5.77, 5.77 and 5.77, while for coated semen, the pH values were adjusted to 6.00, 5.86, 5.74, 5.72, 5.70, 5.69 and 5.67 on days 2, 4, 6, 8, 10, 12 and 14 of storage, respectively, to mimic the pH values measured during regular semen storage without extender renewal. Sperm motility was measured immediately before each extender renewal. 3. Effects of artificially increasing initial extender pH on sperm motility and membrane integrity, during liquid semen storage. Coated semen was stored in mZAP with initial pH adjusted to 6.04 or 6.25 using 1N HCl. Sperm motility and membrane integrity was examined every 48 h of storage. Experiment 3. Evaluating the effect of the new extender n-mZAP on sperm function and fertilizing potential. 1. Comparison of buffering capacity between mZAP and nmZAP. A titration test was performed to compare the
buffering capacity between the mZAP and n-mZAP extenders. The pH of both mZAP and n-mZAP extenders were first adjusted to 6.5, and then, pH of the extenders was measured as they were titrated with increasing volumes of 5N HCl. 2. Effects of mZAP and n-mZAP on sperm motility, membrane integrity and acrosome intactness. Coated semen was stored in n-mZAP or mZAP with an initial pH 6.25. Sperm motility, membrane integrity, acrosome intactness and semen pH values were examined every 48 h. 3. Effects of n-mZAP on sperm fertilizing potential (AI outcomes). Does were inseminated with coated spermatozoa liquid-stored at 5 ◦ C in n-mZAP (pH 6.25) for 0, 12 or 15 d. Semen samples from each goat were used for insemination as evenly as possible on different days of storage. Because semen usually showed a 90% sperm motility immediately after cooling down to 5 ◦ C, 0.5 mL semen in one storage tube should contain about 8.5 × 108 progressively motile spermatozoa. Thus, a storage tube on day 0 of storage was gently centrifuged (30 ◦ C, 100 × g, 1 min) to recover 0.3 mL precipitate for one insemination. Because semen on day 12 of storage showed a 60% sperm motility, 0.75 mL semen were required to obtain 8.5 × 108 progressively motile spermatozoa. Thus, semen from one and a half tube was centrifuged to get 0.3 mL semen for one insemination. Similarly, semen on day 15 of storage showed a sperm motility of 40%, and thus, 1.1 mL semen from 3 tubes were centrifuged to get 0.3 mL semen containing 8.5 × 108 progressively motile spermatozoa for one insemination. 2.10. Data analysis Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. Percentage data were arc sine transformed and analyzed with ANOVA; a Duncan multiple comparison test was used to locate differences. The software used was Statistics Package for Social Science (SPSS 11.5; SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± S.E.M. and P < 0.05 was considered significant. 3. Results Experiment 1. Changes in semen pH and sperm motility during liquid semen storage under different conditions. 1. Changes in sperm motility and semen pH during liquid storage of coated or non-coated semen. Both sperm motility and semen pH value declined with increasing storage time whether spermatozoa were coated or not (Table 1). A correlation analysis (Fig. 1) indicated that sperm motility was strongly correlated with pH value throughout the storage period of both coated (r = 0.816, P < 0.05) and non-coated (r = 0.884, P < 0.05) semen. Sperm coating significantly extended sperm motility although it did not maintain a more stable pH than non-coating. Sperm motility continued to decline after day 6 of storage when pH value became relatively stable, suggesting that
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Table 1 Changes in sperm motility (% progressively motile spermatozoa* ) and semen pH value during liquid storage in mZAP extender of egg-yolk coated or non-coated spermatozoa. Storage time (days)
Non-coated spermatozoa Sperm motility
pH value
Sperm motility
6.06 ± 6.01 ± 5.97 ± 5.91 ± 5.86 ± 5.82 ± 5.76 ± 5.77 ± 5.81 ± 5.77 ± – – –
90.8 86.1 81.6 76.3 70.3 67.1 65.1 59.9 57.9 51.2 45.2 36.4 27.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.03a 0.03a 0.04b 0.03c 0.036d 0.03e 0.05e 0.03e 0.05e 0.05e 0.05f 0.04ef 0.04f
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. without a common letter in superscripts differ significantly (P < 0.05) within the same column. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
a–j
: Values
0.8a 0.9b 1.2c 0.8d 1.2e 1.0f 1.8g 1.5h 1.8i 1.6j
factors other than pH value impaired sperm motility during the later part of storage. 2. Effects of extender renewal on semen pH and sperm motility during liquid semen storage. Extender renewal significantly improved sperm motility (Table 2). Whereas pH value declined with time without extender renewal, it remained constant when extender was renewed. However, sperm motility continued to decline during the later part of storage with extender renewal. Although a strong correlation (r = 0.816, P < 0.05) was observed between sperm motility and extender pH without extender renewal (Fig. 2), correlation was not observed with extender renewal (r = 0.153, P > 0.05). The results suggested that extender renewal improves sperm motility by maintaining a stable pH and that factors other than pH impaired sperm motility during the later part of semen storage. Experiment 2. Effects of artificial pH adjustments on sperm motility during liquid semen storage. 1. Effects of pH stabilization on sperm motility during liquid semen storage. Stabilizing pH significantly improved sperm motility during storage of non-coated semen, but
0.03a 0.01b 0.03b 0.04c 0.03d 0.04d 0.07e 0.02e 0.02e 0.02e
± ± ± ± ± ± ± ± ± ± ± ± ±
pH value 6.04 6.00 5.92 5.86 5.79 5.74 5.73 5.72 5.71 5.70 5.67 5.69 5.67
1 2 3 4 5 6 7 8 9 10 11 12 13
87.8a ± 81.4 ± 72.2 ± 66.0 ± 58.9 ± 47.2 ± 39.9 ± 32.0 ± 22.2 ± 17.9 ± – – –
Coated spermatozoa
1.6a 1.6b 1.6c 3.3d 2.6e 3.1ef 2.4f 2.5g 4.2g 2.9h 6.2i 6.0j 4.2k
it had a mild effect on the coated semen (Table 3). The results suggested that (a) a stable pH was important for long-term liquid semen storage; and (b) sperm coating increased their tolerance to pH fluctuation. 2. Effects of artificially lowered pH on sperm motility during liquid semen storage. Storage with gradually lowered pH significantly impaired sperm motility in the noncoated semen, but only a mild effect observed on the coated semen (Table 4). The results further confirmed that extender renewal during semen storage improves sperm motility by maintaining a stable pH, and that sperm coating increased their tolerance to pH fluctuation. 3. Effects of artificially increasing initial extender pH on sperm motility and membrane integrity during liquid semen storage. From Day 6 of storage onwards, both sperm motility and membrane integrity were significantly higher in semen stored in pH 6.25 extender than in pH 6.04 extender (Table 5). Experiment 3. Effects of n-mZAP on sperm function and fertilizing potential. 1. Comparison of buffering capacity between mZAP and nmZAP. The n-mZAP extender had a higher buffering
Fig. 1. Analysis on the correlation between sperm motility and pH value during liquid storage of egg-yolk coated or non-coated goat spermatozoa. The analysis indicated that sperm motility was strongly correlated with pH value throughout the storage period in both coated (r = 0.816, P < 0.05) and noncoated (r = 0.884, P < 0.05) spermatozoa. The progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
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Table 2 Effects of extender renewal on sperm motility (% progressively motile spermatozoa* ) and semen pH value during liquid storage of egg-yolk coated spermatozoa in mZAP extender. Storage time (days)
Non-renewal Sperm motility
0 2 4 6 8 10 12 13
91.6 85.7 77.9 67.8 59.2 50.8 34.7 27.3
± ± ± ± ± ± ± ±
0.9aA 1.9bA 3.2cA 3.9dA 2.7eA 2.6fA 4.8gA 5.0hA
Renewal pH value 6.10 6.00 5.86 5.74 5.71 5.7 5.7 5.67
± ± ± ± ± ± ± ±
Sperm motility
0.01a 0.02b 0.04c 0.04d 0.02de 0.04de 0.06de 0.05e
90.8 88.2 82.1 75.9 66.9 58.2 45.6 40.2
± ± ± ± ± ± ± ±
1.2aA 1.1abA 1.9bB 2.9cB 3.0dB 2.0eB 4.5fB 3.7fB
pH value 6.08 6.00 5.98 5.98 6.00 5.98 6.02 6.01
± ± ± ± ± ± ± ±
0.01a 0.03b 0.02b 0.03b 0.01b 0.02b 0.01b 0.02b
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a–h : Values without a common letter in superscripts differ significantly (P < 0.05) within the same column. A, B : Sperm motility values with a different letter in superscripts differ significantly (P < 0.05) within the same row. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
Fig. 2. Analysis on the correlation between sperm motility and pH value during liquid semen storage of egg-yolk coated goat spermatozoa with or without extender renewal. The correlation analysis indicated that whereas a strong correlation (r = 0.816, P < 0.05) was observed between sperm motility and pH value without extender renewal, no marked correlation was observed with extender renewal (r = 0.153, P > 0.05). The progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
capacity and maintained a more stable pH condition than the mZAP extender did (Fig. 3). 2. Effects of mZAP and n-mZAP on sperm motility, membrane integrity and acrosome intactness. Semen pH, sperm motility, membrane integrity and acrosome intactness were similar between the two extenders until day 12 of storage when they became significantly lower in mZAP than in n-mZAP (Table 6). The results indicated that nmZAP sustained better sperm quality by maintaining a more stable pH than mZAP did.
3. Effects of n-mZAP on sperm fertilizing potential (AI outcomes). After AI, pregnancy rates and kids number per kidding doe did not differ between semen on day 0 and semen on day 12 of storage (Table 7). Pregnancy rates decreased significantly after semen was stored for 15 d. It should be mentioned that these pregnancy rates were achieved by adjusting the sperm concentration so that a consistent number of motile spermatozoa were inseminated. Thus, goat semen after liquid storage for 12 d in n-mZAP produced pregnancy and kidding rates similar
Table 3 Effects of stabilizing semen pH on sperm motility (% progressively motile spermatozoa* ) during liquid storage in mZAP extender of egg-yolk coated or non-coated spermatozoa. Storage time (days)
Non-coated spermatozoa Non-stabilizing
2 4 6 8 10 12 14
87.7 64.5 50.9 34.2 18.6 0.0 0.0
± ± ± ± ± ± ±
0.7aA 1.1bA 1.3cA 0.8dA 0.9eA 0.0fA 0.0fA
Coated spermatozoa Stabilizing 83.3 71.0 57.8 43.0 25.2 4.3 0.0
± ± ± ± ± ± ±
0.7aA 1.1bB 0.8cB 1.3dB 1.2eB 0.6fB 0.0gA
Non-stabilizing 86.1 76.3 67.1 59.9 51.2 36.4 18.4
± ± ± ± ± ± ±
1.5aA 3.3bBC 2.1cC 2.5dC 1.9eC 1.9fC 4.4gB
Stabilizing 87.3 79.8 68.2 58.6 54.8 43.7 28.2
± ± ± ± ± ± ±
0.9aA 1.2bC 1.3cC 1.5dC 1.9eC 1.9fD 2.0gC
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a–g : Values without a common letter in superscripts differ significantly (P < 0.05) within the same column. A–D : Values without a common letter in superscripts differ significantly (P < 0.05) within the same row. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
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Table 4 Effects of artificially lowered extender pH on sperm motility (% progressively motile spermatozoa* ) during liquid storage in mZAP of egg-yolk coated or non-coated spermatozoa. Storage time (days)
Non-coated spermatozoa Non-lowered (pH)
2 4 6 8 10 12 14
86.7 72.8 62.9 49.3 40.7 24.6 9.7
± ± ± ± ± ± ±
1.2aA (6.00) 1.3bA (5.97) 1.3cA (6.00) 1.7dA (5.99) 2.1eA (6.00) 2.9fA (6.02) 2.6gA (6.00)
Coated spermatozoa Lowered (pH) 84.2 74.8 52.2 41.9 23.8 14.5 6.9
± ± ± ± ± ± ±
Non-lowered (pH)
1.5aA (6.01) 1.8bA (5.91) 1.3cB (5.82) 2.3dB (5.77) 0.9eB (5.77) 1.0fB (5.77) 1.4gA (5.77)
88.1 82.2 75.4 67.0 58.1 45.6 40.4
± ± ± ± ± ± ±
0.5aA (6.00) 0.8bB (5.98) 1.3cC (5.98) 1.1dD (6.00) 0.8eC (5.98) 1.8fC (6.02) 1.5fB (6.01)
Lowered (pH) 88.2 76.7 72.8 59.3 52.7 46.2 36.9
± ± ± ± ± ± ±
0.7aA (6.00) 1.6bA (5.86) 1.9cC (5.74) 2.0dC (5.72) 2.1eC (5.70) 3.5fC (5.69) 3.9gB (5.67)
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a–g : Values without a common letter in superscripts differ significantly (P < 0.05) within the same column. A–D : Values without a common letter in superscripts differ significantly (P < 0.05) within the same row. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C. Table 5 Sperm motility and membrane integrity (% spermatozoa with membrane intact) during liquid storage of egg-yolk coated spermatozoa in mZAP with the initial pH adjusted to 6.04 or 6.25. Storage time (day)
% Progressively motile spermatozoa* pH = 6.04
2 4 6 8 10 12 14 16
85.0 75.9 69.0 59.8 51.6 35.2 18.1 5.2
± ± ± ± ± ± ± ±
0.8A 1.6A 1.5A 1.3A 2.7A 1.7A 0.8A 1.5A
% Spermatozoa with membrane intact
pH = 6.25 89.7 82.9 75.5 73.5 70.1 66.5 55.2 38.6
± ± ± ± ± ± ± ±
1.5A 0.93B 1.3B 1.1B 1.3B 1.3B 1.2B 1.5B
pH = 6.04 92.1 88.7 78.8 70.6 64.6 53.2 37.1 11.4
± ± ± ± ± ± ± ±
0.3A 1.5A 2.0A 2.0A 3.4A 2.8A 4.2A 2.7A
pH = 6.25 93.6 91.2 85.2 84.9 85.2 74.4 70.2 49.9
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. without a common letter in superscripts differ significantly (P < 0.05) within the same row of motility or membrane integrity. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
to those produced with freshly collected semen following AI. 4. Discussion The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually in either mZAP or n-mZAP extenders. Our correlation analysis revealed a strong correlation between sperm motility and semen pH value during the storage. Furthermore, while increasing initial extender pH
Fig. 3. A titration test that compares the buffering capacity between mZAP and n-mZAP extenders. The pH of both mZAP and n-mZAP extenders were first adjusted to 6.5, and then, pH of the extenders was measured as they were titrated with increasing volumes of 5 N HCl.
A,B
± ± ± ± ± ± ± ±
1.9A 2.2A 4.1B 3.2B 2.7B 3.3B 5.0B 3.3B
: Values
from 6.04 to 6.25 or storage with artificially stabilized pH improved sperm quality, storage with gradually lowered extender pH impaired sperm motility. The data suggested metabolic acidosis impaired sperm function during longterm storage of liquid semen. Both mZAP and n-mZAP contained glucose during long-term storage of liquid goat semen. It is accepted that spermatozoa can metabolize glucose through glycolysis for energy supply (Kamp et al., 1996), producing lactate. In the pig, in the presence of glucose, all visible compartments of the spermatozoon as well as the extender were acidified to pH 6.2 within 20 h (Kamp et al., 2003) from the initial pH 7.2 to 7.5 in freshly ejaculated semen (Johnson et al., 2000). Lactate, and several other permeant weak acids, have been shown to reduce the intracellular pH of bovine spermatozoa and many other types of cells (Acott and Carr, 1984). Although it has been reported that depression of intracellular pH by weak acids can reversibly inhibit sperm motility (Jones and Bavister, 2000), which is good for keeping spermatozoa viable during manipulation or preservation (Johnson et al., 2000), it has been suggested that metabolic acidosis may prevent prolonged storage of semen if no proper buffer is present in extenders. The present results supported this speculation by showing that increasing the initial pH of mZAP from 6.04 to 6.25, or storage in n-mZAP with a higher buffering capacity, significantly extended sperm viability during liquid storage of goat semen. One study also showed that sperm motility
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Table 6 Sperm motility, membrane integrity and acrosome intactness during liquid storage of egg-yolk coated spermatozoa in pH 6.25 n-mZAP or mZAP. Storage time (days)
Extenders
pH value
2
n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP
6.25 6.24 6.15 6.12 6.11 6.03 6.05 5.98 6.01 5.90 5.99 5.88 5.93 5.81
10 12 14 16 18 20
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.04a 0.06a 0.04a 0.07a 0.03b 0.05a 0.02b 0.03a 0.09b 0.04a 0.05b 0.04a 0.03b
% Progressively motile spermatozoa* 88.6 89.1 69.6 64.3 67.4 56.0 57.4 44.7 38.5 33.0 22.0 16.9 11.9 6.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
% Spermatozoa with membrane intact
0.5a 0.5a 0.9a 1.0a 1.0a 1.4b 1.3a 1.8b 0.9a 1.9a 1.2a 2.0b 1.8a 1.4a
91.8 92.9 77.6 74.6 75.3 68.0 66.7 56.9 51.4 43.3 34.1 28.3 23.2 14.3
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3a 0.5a 1.2a 1.7a 1.0a 1.5b 1.1a 1.8b 1.6a 1.3b 1.4a 2.2b 2.4a 2.1b
% Spermatozoa with acrosome intact 90.4 90.9 76.5 75.1 77.3 69.4 67.8 59.9 53.0 46.0 35.9 31.3 23.9 18.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.6a 0.4a 1.1a 1.1a 1.2a 1.3b 1.0a 1.6b 1.4a 1.1b 2.2a 1.3a 2.4a 1.8a
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a,b : In the same column, values with different letters in superscripts differ significantly (P < 0.05) on the same day of semen storage. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
decreased significantly when the initial extender pH was reduced from 6.04 to 5.54 (Xu et al., 2009). Exposure to mild acidity acidifies the intracellular pH of human spermatozoa and is rapidly spermicidal (Olmsted et al., 2000). One study found that sperm motility, membrane integrity and acrosomal intactness were significantly higher when extender was renewed at 48-h intervals than when it was not renewed during goat semen storage (Zhao et al., 2009). Extender renewal could have improved sperm function by stabilizing pH, removing detrimental metabolites, or by supplying new nutrients. The present results showed that whereas pH value declined with time without extender renewal, it remained constant when semen was stored with regular extender renewal. When extender renewal was conducted using extenders with artificially lowered pH, however, motility was significantly impaired, particularly in non-coated spermatozoa. The results suggested that extender renewal improved sperm quality by removing acidic metabolites and maintaining a stable pH. It is also possible that renewal of the extender eliminated potential damaging entities such as degradation products from degenerating spermatozoa. The present results demonstrated that sperm coating with yolk improved motility by increasing their tolerance to pH decline during liquid semen storage. Yolk is commonly found in semen extenders to preserve spermatozoa against cold shock during the freeze–thaw process. Although the precise mechanism by which yolk protects
spermatozoa is not quite clear, many authors have proposed that the low density proteins (LDL) contained in the yolk could be largely responsible for sperm protection (Evans et al., 1968; Pace and Graham, 1974; Quinn et al., 1980). In fact, some authors have suggested that LDL could adhere to cell membrane during the freeze-thaw process, thus preserving sperm membranes (Foulkes, 1977; Polge, 1980; Graham and Foote, 1987). Furthermore, yolk may also have antioxidant effects during sperm cryopreservation (Alvarez-Rodríguez et al., 2013). Thus, the present results have added a new function to yolk that increases sperm tolerance to pH fluctuations during semen storage. In this study, when semen was stored without extender renewal, the pH value became relatively stable from day 6 of storage onwards; however, sperm motility continued to decline thereafter. Similarly, although pH was maintained constant with extender renewal, sperm motility declined particularly during the later part of the storage. The data suggested that factors other than pH impaired sperm quality particularly during the later part of semen storage. Studies have demonstrated that human spermatozoa are capable of generating reactive oxygen species (ROS), and ROS production in semen has been associated with loss of sperm motility, decreased capacity for sperm-oocyte fusion and loss of fertility (Griveau and Le Lannou, 1997). Lipid peroxidation is significantly accelerated in populations of defective spermatozoa exhibiting high levels of ROS production or in normal cells stimulated to produce oxygen
Table 7 Rates of pregnancy, abortion and kidding after AI using goat semen liquid stored in n-mZAP for different days.* Storage time (days)
% Pregnancy (pregnant/ inseminated does)
% Abortion (aborted/pregnant does recorded)
Average number of kids (kids/does recorded)
0 12 15
79.3 (23/29)a 65.7 (25/38)a 20.0 (5/25)b
16.7 (3/18)a 13.6 (3/22)a 0 (0/2)a
1.93 ± 0.18 (29/15)a 1.78 ± 0.13 (34/19)a 2.00 ± 0.00 (4/2)a
a,b : In the same column, values with different letters in superscripts differ significantly (P < 0.05). Percentage data for pregnancy and abortion rates were analyzed with Chi-squarer (x2 ) test, while data for live kids per pregnant doe were analyzed with ANOVA as described in Section 2. * These pregnancy rates were achieved by adjusting the sperm concentration so that a consistent number of motile spermatozoa were inseminated.
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radicals (Aitken et al., 1989). Furthermore, our recent study observed a remarkable rise in the oxidative stress index (OSI) on day 14 of goat semen storage in n-mZAP (data not shown). In this study, with the same numbers of progressively motile spermatozoa inseminated, pregnancy rates were significantly lower when semen was stored for 15 d than when semen was stored for 0 or 12 d. While sperm motility, membrane integrity and acrosome intactness were similar between day 10 and day 12, they decreased by about 10% between day 12 and day 14 of semen storage in n-mZAP (Table 6). Similarly, although OSI did not differ between day 4 and day 12, it increased significantly on day 14 of goat semen storage in n-mZAP (data not shown). Sperm motility, while an essential feature of healthy spermatozoa, is not necessarily indicative of fertilizing capacity (Hafez, 1992). Normal spermatozoa lose fertilizing ability before they lose motility. In summary, the present results showed that during long-term liquid storage of goat semen, a gradual decline in pH was closely correlated with a decrease in sperm motility. Extender renewal improved sperm motility by maintaining a stable pH, while sperm coating improved motility by increasing their tolerance to pH decline. With increased initial pH and higher buffering capacity, n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than mZAP did. Goat semen liquid stored in n-mZAP for 12 d produced pregnancy and kidding rates similar to those obtained with freshly collected semen following AI with the sperm concentration adjusted so that a consistent number of motile spermatozoa were inseminated. The data are important for protocol optimization for long-term semen storage not only in the goat but also in other species. Conflicts of interest We have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled: Effects of pH during liquid storage of goat semen on sperm viability and fertilizing potential. Acknowledgements This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (No. 31272444) and the Animal Breed Improvement Program of Shandong Province. References Acott, T.S., Carr, D.W., 1984. Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of pH and a quiescence factor. Biol. Reprod. 30, 926–935. Aitken, R.J., Clarkson, J.S., Fishel, S., 1989. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 41, 183–197. Alvarez-Rodríguez, M., Alvarez, M., Anel-López, L., Martínez-Rodríguez, C., Martínez-Pastor, F., Borragan, S., Anel, L., de Paz, P., 2013. The antioxidant effects of soybean lecithin- or low-density
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lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa. Reprod. Fertil. Dev. 25, 1185–1193. Corteel, J.M., 1981. Collection processing and artificial insemination of goat semen. In: Gall, C. (Ed.), Goat Production. Academic Press, London, pp. 171–191. De Pauw, I.M., Van Soom, A., Maes, D., Verberckmoes, S., de Kruif, A., 2003. Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa. Theriogenology 59, 1109–1122. Evans, R.J., Bandemer, S.L., Davidson, J.A., Heinlein, K., Vaghefi, S.S., 1968. Binding of lipid to protein in the low-density lipoprotein from the hen’s egg. Biochim. Biophys. Acta 164, 566–574. Foulkes, J.A., 1977. The separation of lipoproteins from egg yolk and their effect on the motility and integrity of bovine spermatozoa. J. Reprod. Fertil. 49, 277–284. Gottardi, L., Brunei, L., Zanelli, L., 1980. New dilution media for artificial insemination in the pig. In: 9th International Congress of Animal Reproduction and Artificial Insemination, Madrid, vol. 5, pp. 49–53. Graham, J.K., Foote, R.H., 1987. Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing. Cryobiology 24, 42–52. Griveau, J.F., Le Lannou, D., 1997. Reactive oxygen species human spermatozoa: physiology and pathology. Int. J. Androl. 20, 61–69. Hafez, E.S.E., 1992. Semen evaluation. In: Hafez, E.S.E. (Ed.), Reproduction in Farm Animals. , 6th ed. Lea & Febiger, Philadelphia, pp. 405–423. Harrison, R.A., White, I.G., 1972. Glycolytic enzymes in the spermatozoa and cytoplasmic droplets of bull, boar and ram, and their leakage after shock. J. Reprod. Fertil. 30, 105–115. Johnson, L.A., Weitze, K.F., Fiser, P., Maxwell, W.M., 2000. Storage of boar semen. Anim. Reprod. Sci. 62, 143–172. Johnston, S.D., McGowan, M.R., Phillips, N.J., O’Callaghan, P., 2000. Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa. J. Reprod. Fertil. 118, 273–281. Jones, J.M., Bavister, B.D., 2000. Acidification of intracellular pH in bovine spermatozoa suppresses motility and extends viable life. J. Androl. 21, 616–624. Kamp, G., Büsselmann, G., Jones, N., Wiesner, B., Lauterwein, J., 2003. Energy metabolism and intracellular pH in boar spermatozoa. Reproduction 126, 517–525. Kamp, G., Busselmann, G., Lauterwein, J., 1996. Spermatozoa: models for studying regulatory aspects of energy metabolism. Experientia 52, 487–494. Khalifa, T.A., El-Saidy, B.E., 2006. Pellet-freezing of Damascus goat semen in a chemically defined extender. Anim. Reprod. Sci. 93, 303–315. Kundu, C.N., Chakrabarty, J., Dutta, P., Bhattacharyya, D., Ghosh, A., Majumder, G.C., 2002. Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium. Reproduction 123, 907–913. Kundu, C.N., Chakraborty, J., Dutta, P., Bhattacharyya, D., Ghosh, A., Majumder, G.C., 2000. Development of a simple sperm cryopreservation model using a chemically defined medium and goat cauda epididymal spermatozoa. Cryobiology 40, 117–125. Larson, J.L., Miller, D.J., 1999. Simple histochemical stain for acrosomes on sperm from several species. Mol. Reprod. Dev. 52, 445–449. Leboeuf, B., Guillouet, P., Batellier, F., Bernelas, D., Bonné, J.L., Forgerit, Y., Renaud, G., Magistrini, M., 2003. Effect of native phosphocaseinate on the in vitro preservation of fresh semen. Theriogenology 60, 867–877. Leboeuf, B., Restall, B., Salamon, S., 2000. Production and storage of goat semen for artificial insemination. Anim. Reprod. Sci. 62, 113–141. Martínez, M.F., Bosch, P., Bosch, R.A., 1998. Determination of early pregnancy and embryonic growth in goats by transrectal ultrasound scanning. Theriogenology 49, 1555–1565. Maxwell, W.M., Salamon, S., 1993. Liquid storage of ram semen: a review. Reprod. Fertil. Dev. 5, 613–638. Olmsted, S.S., Dubin, N.H., Cone, R.A., Moench, T.R., 2000. The rate at which human sperm are immobilized and killed by mild acidity. Fertil. Steril. 73, 687–693. Pace, M.M., Graham, E.F., 1974. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39, 1144–1149. Paulenz, H., Soltun, K., Adnoy, T., Berg, K.A., Soderquist, L., 2005. Effect of different extenders on sperm viability of buck semen stored at room temperature. Small Rumin. Res. 59, 89–94. Polge, C., 1980. Freezing of spermatozoa. In: Ashwood-Smith, M.J., Farrant, J. (Eds.), Low Temperature Preservation in Medicine and Biology. Tunbridge Wells, Kent, Pitman Medical, pp. 45–64. Quinn, P.J., Chow, P.Y., White, I.G., 1980. Evidence that phospholipid protects ram spermatozoa from cold shock at a plasma membrane site. J. Reprod. Fertil. 60, 403–407.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effects of pH during liquid storage of goat semen on sperm viability and fertilizing potential Chang-He Liu 1 , Hai-Bo Dong 1 , Dong-Li Ma 1 , You-Wei Li, Dong Han, Ming-Jiu Luo, Zhong-Le Chang ∗ , Jing-He Tan ∗ College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an City 271018, P.R. China
a r t i c l e
i n f o
Article history: Received 29 August 2015 Received in revised form 16 October 2015 Accepted 6 November 2015 Available online 10 November 2015 Keywords: Liquid semen storage Extender pH Sperm motility Fertilizing potential Goat
a b s t r a c t A specific problem in goat semen preservation is the detrimental effect of seminal plasma on sperm viability in extenders containing yolk or milk. Thus, the use of chemically defined extenders will have obvious advantages. Although previous studies indicate that the initial pH of an extender is crucial to sustain high sperm motility, changes in extender pH during long-term semen storage have not been observed. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus important for protocol optimization for long-term liquid semen preservation. The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually, and a strong correlation was observed between the two. Whereas increasing the initial extender pH from 6.04 to 6.25 or storage with stabilized pH improved, storage with artificially lowered pH impaired sperm motility. Extender renewal improved sperm motility by maintaining a stable pH. Sperm coating with chicken (Gallus gallus) egg yolk improved motility by increasing tolerance to pH decline. A new extender (n-mZAP) with a higher buffering capacity was formulated, and n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than the currently used mZAP extender did. Goat semen liquid-stored for 12 d in n-mZAP produced pregnancy and kidding rates similar to those obtained with freshly collected semen following artificial insemination. In conclusion, maintenance of a stable pH during liquid semen storage dramatically improved sperm viability and fertilizing potential. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Liquid-stored semen can be an alternative to frozenthawed semen for artificial insemination (AI), because semen cryopreservation is an expensive process. However, although much research has already been conducted
∗ Corresponding authors. E-mail addresses: [email protected] (Z.-L. Chang), [email protected] (J.-H. Tan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.anireprosci.2015.11.011 0378-4320/© 2015 Elsevier B.V. All rights reserved.
to prolong the in vitro viability and fertilizing potential of stored liquid semen, limited improvements have been achieved in different species (Weitze, 1990; De Pauw et al., 2003; Leboeuf et al., 2003). A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of the spermatozoa in extenders containing egg yolk or milk (Leboeuf et al., 2000). The use of milk- or yolk-containing extenders in this species therefore requires the removal of the seminal plasma by washing before semen dilution. Because washing is a complex and time-consuming process, and it causes damage (Harrison and White, 1972) and some loss (Corteel,
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1981) of spermatozoa, the use of chemically defined extenders will have obvious advantages in liquid storage of goat semen. However, reports on the liquid storage of goat semen in chemically defined extenders which contain neither milk nor yolk are few (Leboeuf et al., 2003; Paulenz et al., 2005); so are reports on cryopreservation of goat spermatozoa in such chemically defined media (Kundu et al., 2000, 2002; Khalifa and El-Saidy, 2006). One study (Xu et al., 2009) has shown that the self-made mZAP extender, formulated based on the Zorlesco extender (Gottardi et al., 1980) and the Androhep® extender (Weitze, 1990) used for liquid storage of porcine semen, performed the best among the extenders tested. A forward sperm motility of over 30% was maintained for 9 d and successful pregnancies were induced after AI with semen stored for 7 d when goat semen was diluted and stored in the mZAP extender. Another study (Zhao et al., 2009) examined effects of semen cooling velocity, sperm coating and extender renewal on sperm viability during goat semen storage. With the optimized protocol, a sperm motility of 48% was maintained for 13 d, and an in vitro-fertilizing potential similar to that of fresh semen was maintained for 11 d. The pH of freshly ejaculated boar semen is between 7.2 and 7.5 (Johnson et al., 2000). When glucose was present in the extender, all visible compartments of the boar spermatozoa as well as the extender were acidified to pH 6.2 within 20 h (Kamp et al., 2003). Although it is recognized that a lower pH can reduce sperm motility and metabolic activity, which is good for keeping spermatozoa viable during manipulation or preservation (Johnson et al., 2000), metabolic acidosis may prevent prolonged storage if no proper buffer is present in the extenders. Thus, our previous study demonstrated that whereas 6.04 was the most suitable pH for liquid storage of goat semen, sperm viability decreased significantly when semen was stored at either pH 6.61 or pH 5.54 (Xu et al., 2009). While these results and those from other studies (Maxwell and Salamon, 1993; Johnston et al., 2000) indicate that the initial pH of an extender is crucial to sustain high sperm motility during semen manipulation or storage, changes in semen pH during longterm storage have not been reported. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus of great importance for protocol optimization for long-term liquid semen preservation. The objective of this study was to observe the effect of pH during liquid semen storage in chemically defined extenders on goat sperm viability and fertilizing potential. Experiment 1 monitored and correlated semen pH and sperm motility changes during the storage of coated or non-coated spermatozoa with or without extender renewal. In experiment 2, effects of artificially adjusting semen pH on sperm motility were observed. The adjustments made included stabilizing or lowering pH during storage and increasing the initial extender pH. In experiment 3, a new extender that possesses a higher buffering capacity was developed and its effect on sperm function and fertilizing potential (AI outcomes) was evaluated.
2. Materials and methods 2.1. Ethics statement The experimental procedures were approved by the Animal Care and Use Committee of the Shandong Agricultural University P. R. China (Permit number: SDAUA-2001-001). All chemicals and media used were purchased from Sigma Chemical Co (St. Louis, MO, USA), unless specified otherwise. 2.2. Animals and semen collection The study was conducted at the Animal Station of the Shandong Agricultural University, Shandong Province (122◦ to 114 ◦ E; 34◦ to 38 ◦ N) of China. Male goats were kept in sheltered pens separated from females, and fed hay and concentrate, with water available ad libitum. The Lubei White male goats (n = 5) used in this study were of meat breed. Male goats aged between 2 and 4 yr were trained to ejaculate into an artificial vagina at a doe mount. Semen collection was scheduled to be at 3-d intervals. The ejaculate was collected into a pre-warmed empty tube. 2.3. Extenders The mZAP extender, which contained 63.9 mM glucose, 80.6 mM fructose, 39.8 mM sodium citrate, 5.1 mM ethylenediaminetetraacetic acid (EDTA), 14.9 mM NaHCO3 , 37.8 mM Hepes, 0.25 g/100 mL polyvinyl alcohol (PVA), 5000 IU/100 mL penicillin and 0.1 g/100 mL streptomycin, was prepared as reported previously (Xu et al., 2009). The new mZAP (n-mZAP) extender contained the same constituents as the mZAP did except that NaHCO3 and Hepes in the mZAP extender was replaced with 2-morpholinoethanesulfonic acid (MES) as buffers. The pH of the extenders was adjusted to 6.04 with 1N HCl unless specified otherwise. 2.4. Sperm coating and semen dilution and packaging In experiments not involving sperm coating, ejaculates of original volume were 1:10 diluted with pre-warmed (35 ◦ C) extender. In the sperm-coating experiment, ejaculates were 1:1 diluted with the extender supplemented with 20% chicken (Gallus gallus) egg yolk. Within 5 min, the coated ejaculates were centrifuged at 30 ◦ C for 10 min at 200 × g. Following removal of the supernatant, the coated ejaculates were 1:10 diluted with pre-warmed extender. Both the non-coated and coated ejaculates that had been 1:10 diluted were packaged in 1.5-mL microfuge tubes (0.5 mL semen per tube) and the tubes were then placed in a water bath immediately after packaging and maintained at 35 ◦ C before cooling and storage. 2.5. Semen cooling and storage After packaging, the semen was cooled to and stored at 5 ◦ C in an incubator. Cooling down to 5 ◦ C was achieved by incubating at 5 ◦ C the storage tubes with extended semen
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in a 200-mL pre-warmed (30 ◦ C) water bath as described in our previous study (Zhao et al., 2009). 2.6. Semen pH measurement and sperm quality assessment After semen samples for sperm quality assessment were taken out, the rest semen in the storage tube was centrifuged at 4 ◦ C for 10 min at 500 × g. At the end of the centrifugation, supernatant was collected for pH measurement. The pH was measured using a Denver Instrument pH Meter (UB-7, Denver Instrument, 5 Orville Dr. Bohemia, NY 11716) equipped with a 15-L probe. Sperm motility was analyzed by a computer-assisted sperm analyzer (CASA) system (Sperm Class Analyzer, Microptic SL, Barcelona, Spain). For analysis, 100 L semen samples were mixed with 200 L pre-warmed (37 ◦ C) mZAP extender. Then, 5 L of the sperm suspension (about 60–120 million cells/mL) was placed on a pre-warmed microscope slide, and overlaid with a 22 mm2 coverslip. The slide was maintained at 37 ◦ C during analysis by a heated slide warmer (TOKAT HIT MATS-U55R30, TOKAI HIT CO., LTD, Japan) and observed with the Nikon phase contrast microscope (Eclipse E-200, Nikon Co., Tokyo, Japan) at 200 × magnification. Several fields of view were captured and at least 1000 spermatozoa were counted in each analysis. With respect to the setting parameters, the system has a specific setup for goat sperm evaluation. It was set up as follows: VCL (m/s): 10 < slow < 40 < medium < 75 < rapid; VAP (m/s): 10 < slow < 40 < medium < 75 < rapid; LIN: circular < 50%; Progressivity: 80% of STR. The system issued a sperm motility classification according to the WHO standard parameters as follows. Type A, Rapid progressive; Type B, Slow progressive; Type C, Non- progressive; and Type D, Immotile. The four categories are expressed in percentages. Percentage of motile spermatozoa: A+B+C; Percentage of progressive motile spermatozoa: A+B. In this study, the term “motility” was used to refer percentage of progressive motile spermatozoa. Membrane integrity of spermatozoa was evaluated by the osmotic resistance test (ORT) (Revell and Mrodeb, 1994; Zhou et al., 2004). Briefly, 25 L of semen was added to 200 L of pre-warmed hypo-osmotic solution and mixed thoroughly. After incubation for 45 min at 38.5 ◦ C, 300 L of 2% glutaraldehyde was added. Then, 10 L of the sperm mixture was placed on a hemocytometer and examined under a phase-contrast microscope at a magnification of 400×. A positive response to the test was evident by the coiling of the sperm tail. Both the total number of spermatozoa and the number of spermatozoa with coiled tails in each medium square was counted. The counting was carried out on individual spermatozoa in five to six different medium squares until 200 cells had been counted. Acrosomal intactness was assessed by Coomassie Blue G-250 staining (Larson and Miller, 1999). Briefly, 50 L of semen was added to 1 mL of saline, mixed and centrifuged at 200 × g for 15 s. The pellet was resuspended in 1 mL of paraformaldehyde in PBS and fixed for 30 min. The spermatozoa were washed by centrifugation, and the pellet was resuspended in PBS and spread on a slide for air-drying. The smear was stained for 5 min with 0.22% Coomassie
49
Blue G-250 and washed in water. After air-drying, the smear was observed for sperm acrosomal status. The intact acrosomes were stained intensely blue in color, but the spermatozoa that had lost acrosome integrity lacked Coomassie staining over the acrosomal region. The number of spermatozoa with intact acrosomes and the total number of spermatozoa were counted in different fields. 2.7. Artificial insemination Cycling Lubei White does, aged 2–3 yr, were used. Estrus was checked with a vasectomized goats twice a day at 06:00 and 18:00 h. Females in heat were cervically inseminated with stored semen. The inseminating dose was 0.3 mL semen containing 8.5 × 108 progressively motile spermatozoa. Does that began estrus in the morning were first inseminated in the evening the same day, while those found in heat in the evening were inseminated for the first time in the following morning. The does were inseminated for the second time at 12 h after the first insemination. 2.8. Pregnancy determination via ultrasonography Ultrasonography was performed as reported previously (Martínez et al., 1998). Does were restrained in the standing position in a sqeeze chute. After fecal pellets were removed, conventional ultrasound gel (about 50 mL) was inserted with a syringe in to the rectum to act as a coupling medium between the rectal wall and transducer. The transducer was inserted and manipulated in the rectum by external control of the extension. Each dose was scanned transrectally by using a real-time ultrasound scanner equipped with a 5–7.5–9 MHz alternative linear array transducer (HS-2000, Tokyo, Japan) at 25 d post insemination. Once the embryonic vesicle and heart beat were detected, the doe was considered pregnant. If only the embryonic vesicle was detected, the doe was examined again 10 d later to detect fetus; a doe was considered pregnant only when both the vesicle and fetus were detected. Pregnant does were kept until parturition. 2.9. Experimental design Each treatment was repeated 3 times using ejaculates from three different male goats, and thus, each replicate contained one ejaculate from each male goat. Ejaculates from different goats were processed and stored individually. For example, an ejaculate from Goat A, which had a volume of 1 mL, was packaged and stored in 22 storage tubes each containing 0.5 mL semen. Sperm quality and semen pH on day 0 of storage were examined when the packaged semen was cooled to 5 ◦ C, and they were examined every 24 or 48 h of storage thereafter. Every time, one tube was taken from each treatment for examination of pH and sperm quality. Experiment 1. Monitoring changes in semen pH and sperm motility during liquid semen storage under different conditions 1. Changes in sperm motility and semen pH during liquid storage of coated or non-coated semen. Non-coated and
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coated semen samples were stored in the mZAP extender. Sperm motility and semen pH were examined every 24 h of the storage. 2. Effects of extender renewal on semen pH and sperm motility during liquid semen storage. Coated semen was stored in the mZAP extender, and half of the extender was renewed every 48 h during semen storage. To renew the extender, 0.25 mL of supernatant was removed with a pipette from each storage tube and the same amount of fresh extender that had been pre-cooled to 5 ◦ C was injected into the same tube. The procedure was performed with caution, so as not to disturb the sperm pellet. Sperm motility and extender pH were measured immediately before each extender renewal. Experiment 2. Evaluating the effect of artificial pH adjustments on sperm motility during liquid semen storage. 1. Effects of pH stabilization on sperm motility during liquid semen storage. Non-coated and coated semen were stored in the mZAP extender. Whereas semen in the pH non-stabilizing group was stored undisturbed without any manipulation, the pH of semen in the pH stabilizing group was adjusted to 6.04 everyday during the storage. To stabilize semen pH, one storage tube was taken from each treatment every 24 h of storage. Then, 0.25 mL supernatant was collected from the tube. After pH measurement, the pH of the collected supernatant was adjusted by adding increasing amounts of 1N NaOH. The amount of NaOH used was recorded when the pH reached 6.04 and used for pH adjustment in the remaining tubes of the same treatment on the same day. 2. Effects of artificially lowered pH on sperm motility during liquid semen storage. To store semen with artificially lowered extender pH during storage, an extender renewal system was adopted with half of the extender renewed every 48 h during the storage. While the extender was replaced with fresh regular mZAP (pH 6.04) in the pH non-lowered group, it was renewed using mZAP with pre-adjusted pH values in the pH-lowered group. For non-coated semen, the pH values of mZAP extender used for renewal were adjusted to 6.01, 5.91, 5.82, 5.77, 5.77, 5.77 and 5.77, while for coated semen, the pH values were adjusted to 6.00, 5.86, 5.74, 5.72, 5.70, 5.69 and 5.67 on days 2, 4, 6, 8, 10, 12 and 14 of storage, respectively, to mimic the pH values measured during regular semen storage without extender renewal. Sperm motility was measured immediately before each extender renewal. 3. Effects of artificially increasing initial extender pH on sperm motility and membrane integrity, during liquid semen storage. Coated semen was stored in mZAP with initial pH adjusted to 6.04 or 6.25 using 1N HCl. Sperm motility and membrane integrity was examined every 48 h of storage. Experiment 3. Evaluating the effect of the new extender n-mZAP on sperm function and fertilizing potential. 1. Comparison of buffering capacity between mZAP and nmZAP. A titration test was performed to compare the
buffering capacity between the mZAP and n-mZAP extenders. The pH of both mZAP and n-mZAP extenders were first adjusted to 6.5, and then, pH of the extenders was measured as they were titrated with increasing volumes of 5N HCl. 2. Effects of mZAP and n-mZAP on sperm motility, membrane integrity and acrosome intactness. Coated semen was stored in n-mZAP or mZAP with an initial pH 6.25. Sperm motility, membrane integrity, acrosome intactness and semen pH values were examined every 48 h. 3. Effects of n-mZAP on sperm fertilizing potential (AI outcomes). Does were inseminated with coated spermatozoa liquid-stored at 5 ◦ C in n-mZAP (pH 6.25) for 0, 12 or 15 d. Semen samples from each goat were used for insemination as evenly as possible on different days of storage. Because semen usually showed a 90% sperm motility immediately after cooling down to 5 ◦ C, 0.5 mL semen in one storage tube should contain about 8.5 × 108 progressively motile spermatozoa. Thus, a storage tube on day 0 of storage was gently centrifuged (30 ◦ C, 100 × g, 1 min) to recover 0.3 mL precipitate for one insemination. Because semen on day 12 of storage showed a 60% sperm motility, 0.75 mL semen were required to obtain 8.5 × 108 progressively motile spermatozoa. Thus, semen from one and a half tube was centrifuged to get 0.3 mL semen for one insemination. Similarly, semen on day 15 of storage showed a sperm motility of 40%, and thus, 1.1 mL semen from 3 tubes were centrifuged to get 0.3 mL semen containing 8.5 × 108 progressively motile spermatozoa for one insemination. 2.10. Data analysis Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. Percentage data were arc sine transformed and analyzed with ANOVA; a Duncan multiple comparison test was used to locate differences. The software used was Statistics Package for Social Science (SPSS 11.5; SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± S.E.M. and P < 0.05 was considered significant. 3. Results Experiment 1. Changes in semen pH and sperm motility during liquid semen storage under different conditions. 1. Changes in sperm motility and semen pH during liquid storage of coated or non-coated semen. Both sperm motility and semen pH value declined with increasing storage time whether spermatozoa were coated or not (Table 1). A correlation analysis (Fig. 1) indicated that sperm motility was strongly correlated with pH value throughout the storage period of both coated (r = 0.816, P < 0.05) and non-coated (r = 0.884, P < 0.05) semen. Sperm coating significantly extended sperm motility although it did not maintain a more stable pH than non-coating. Sperm motility continued to decline after day 6 of storage when pH value became relatively stable, suggesting that
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Table 1 Changes in sperm motility (% progressively motile spermatozoa* ) and semen pH value during liquid storage in mZAP extender of egg-yolk coated or non-coated spermatozoa. Storage time (days)
Non-coated spermatozoa Sperm motility
pH value
Sperm motility
6.06 ± 6.01 ± 5.97 ± 5.91 ± 5.86 ± 5.82 ± 5.76 ± 5.77 ± 5.81 ± 5.77 ± – – –
90.8 86.1 81.6 76.3 70.3 67.1 65.1 59.9 57.9 51.2 45.2 36.4 27.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.03a 0.03a 0.04b 0.03c 0.036d 0.03e 0.05e 0.03e 0.05e 0.05e 0.05f 0.04ef 0.04f
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. without a common letter in superscripts differ significantly (P < 0.05) within the same column. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
a–j
: Values
0.8a 0.9b 1.2c 0.8d 1.2e 1.0f 1.8g 1.5h 1.8i 1.6j
factors other than pH value impaired sperm motility during the later part of storage. 2. Effects of extender renewal on semen pH and sperm motility during liquid semen storage. Extender renewal significantly improved sperm motility (Table 2). Whereas pH value declined with time without extender renewal, it remained constant when extender was renewed. However, sperm motility continued to decline during the later part of storage with extender renewal. Although a strong correlation (r = 0.816, P < 0.05) was observed between sperm motility and extender pH without extender renewal (Fig. 2), correlation was not observed with extender renewal (r = 0.153, P > 0.05). The results suggested that extender renewal improves sperm motility by maintaining a stable pH and that factors other than pH impaired sperm motility during the later part of semen storage. Experiment 2. Effects of artificial pH adjustments on sperm motility during liquid semen storage. 1. Effects of pH stabilization on sperm motility during liquid semen storage. Stabilizing pH significantly improved sperm motility during storage of non-coated semen, but
0.03a 0.01b 0.03b 0.04c 0.03d 0.04d 0.07e 0.02e 0.02e 0.02e
± ± ± ± ± ± ± ± ± ± ± ± ±
pH value 6.04 6.00 5.92 5.86 5.79 5.74 5.73 5.72 5.71 5.70 5.67 5.69 5.67
1 2 3 4 5 6 7 8 9 10 11 12 13
87.8a ± 81.4 ± 72.2 ± 66.0 ± 58.9 ± 47.2 ± 39.9 ± 32.0 ± 22.2 ± 17.9 ± – – –
Coated spermatozoa
1.6a 1.6b 1.6c 3.3d 2.6e 3.1ef 2.4f 2.5g 4.2g 2.9h 6.2i 6.0j 4.2k
it had a mild effect on the coated semen (Table 3). The results suggested that (a) a stable pH was important for long-term liquid semen storage; and (b) sperm coating increased their tolerance to pH fluctuation. 2. Effects of artificially lowered pH on sperm motility during liquid semen storage. Storage with gradually lowered pH significantly impaired sperm motility in the noncoated semen, but only a mild effect observed on the coated semen (Table 4). The results further confirmed that extender renewal during semen storage improves sperm motility by maintaining a stable pH, and that sperm coating increased their tolerance to pH fluctuation. 3. Effects of artificially increasing initial extender pH on sperm motility and membrane integrity during liquid semen storage. From Day 6 of storage onwards, both sperm motility and membrane integrity were significantly higher in semen stored in pH 6.25 extender than in pH 6.04 extender (Table 5). Experiment 3. Effects of n-mZAP on sperm function and fertilizing potential. 1. Comparison of buffering capacity between mZAP and nmZAP. The n-mZAP extender had a higher buffering
Fig. 1. Analysis on the correlation between sperm motility and pH value during liquid storage of egg-yolk coated or non-coated goat spermatozoa. The analysis indicated that sperm motility was strongly correlated with pH value throughout the storage period in both coated (r = 0.816, P < 0.05) and noncoated (r = 0.884, P < 0.05) spermatozoa. The progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
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Table 2 Effects of extender renewal on sperm motility (% progressively motile spermatozoa* ) and semen pH value during liquid storage of egg-yolk coated spermatozoa in mZAP extender. Storage time (days)
Non-renewal Sperm motility
0 2 4 6 8 10 12 13
91.6 85.7 77.9 67.8 59.2 50.8 34.7 27.3
± ± ± ± ± ± ± ±
0.9aA 1.9bA 3.2cA 3.9dA 2.7eA 2.6fA 4.8gA 5.0hA
Renewal pH value 6.10 6.00 5.86 5.74 5.71 5.7 5.7 5.67
± ± ± ± ± ± ± ±
Sperm motility
0.01a 0.02b 0.04c 0.04d 0.02de 0.04de 0.06de 0.05e
90.8 88.2 82.1 75.9 66.9 58.2 45.6 40.2
± ± ± ± ± ± ± ±
1.2aA 1.1abA 1.9bB 2.9cB 3.0dB 2.0eB 4.5fB 3.7fB
pH value 6.08 6.00 5.98 5.98 6.00 5.98 6.02 6.01
± ± ± ± ± ± ± ±
0.01a 0.03b 0.02b 0.03b 0.01b 0.02b 0.01b 0.02b
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a–h : Values without a common letter in superscripts differ significantly (P < 0.05) within the same column. A, B : Sperm motility values with a different letter in superscripts differ significantly (P < 0.05) within the same row. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
Fig. 2. Analysis on the correlation between sperm motility and pH value during liquid semen storage of egg-yolk coated goat spermatozoa with or without extender renewal. The correlation analysis indicated that whereas a strong correlation (r = 0.816, P < 0.05) was observed between sperm motility and pH value without extender renewal, no marked correlation was observed with extender renewal (r = 0.153, P > 0.05). The progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
capacity and maintained a more stable pH condition than the mZAP extender did (Fig. 3). 2. Effects of mZAP and n-mZAP on sperm motility, membrane integrity and acrosome intactness. Semen pH, sperm motility, membrane integrity and acrosome intactness were similar between the two extenders until day 12 of storage when they became significantly lower in mZAP than in n-mZAP (Table 6). The results indicated that nmZAP sustained better sperm quality by maintaining a more stable pH than mZAP did.
3. Effects of n-mZAP on sperm fertilizing potential (AI outcomes). After AI, pregnancy rates and kids number per kidding doe did not differ between semen on day 0 and semen on day 12 of storage (Table 7). Pregnancy rates decreased significantly after semen was stored for 15 d. It should be mentioned that these pregnancy rates were achieved by adjusting the sperm concentration so that a consistent number of motile spermatozoa were inseminated. Thus, goat semen after liquid storage for 12 d in n-mZAP produced pregnancy and kidding rates similar
Table 3 Effects of stabilizing semen pH on sperm motility (% progressively motile spermatozoa* ) during liquid storage in mZAP extender of egg-yolk coated or non-coated spermatozoa. Storage time (days)
Non-coated spermatozoa Non-stabilizing
2 4 6 8 10 12 14
87.7 64.5 50.9 34.2 18.6 0.0 0.0
± ± ± ± ± ± ±
0.7aA 1.1bA 1.3cA 0.8dA 0.9eA 0.0fA 0.0fA
Coated spermatozoa Stabilizing 83.3 71.0 57.8 43.0 25.2 4.3 0.0
± ± ± ± ± ± ±
0.7aA 1.1bB 0.8cB 1.3dB 1.2eB 0.6fB 0.0gA
Non-stabilizing 86.1 76.3 67.1 59.9 51.2 36.4 18.4
± ± ± ± ± ± ±
1.5aA 3.3bBC 2.1cC 2.5dC 1.9eC 1.9fC 4.4gB
Stabilizing 87.3 79.8 68.2 58.6 54.8 43.7 28.2
± ± ± ± ± ± ±
0.9aA 1.2bC 1.3cC 1.5dC 1.9eC 1.9fD 2.0gC
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a–g : Values without a common letter in superscripts differ significantly (P < 0.05) within the same column. A–D : Values without a common letter in superscripts differ significantly (P < 0.05) within the same row. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
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Table 4 Effects of artificially lowered extender pH on sperm motility (% progressively motile spermatozoa* ) during liquid storage in mZAP of egg-yolk coated or non-coated spermatozoa. Storage time (days)
Non-coated spermatozoa Non-lowered (pH)
2 4 6 8 10 12 14
86.7 72.8 62.9 49.3 40.7 24.6 9.7
± ± ± ± ± ± ±
1.2aA (6.00) 1.3bA (5.97) 1.3cA (6.00) 1.7dA (5.99) 2.1eA (6.00) 2.9fA (6.02) 2.6gA (6.00)
Coated spermatozoa Lowered (pH) 84.2 74.8 52.2 41.9 23.8 14.5 6.9
± ± ± ± ± ± ±
Non-lowered (pH)
1.5aA (6.01) 1.8bA (5.91) 1.3cB (5.82) 2.3dB (5.77) 0.9eB (5.77) 1.0fB (5.77) 1.4gA (5.77)
88.1 82.2 75.4 67.0 58.1 45.6 40.4
± ± ± ± ± ± ±
0.5aA (6.00) 0.8bB (5.98) 1.3cC (5.98) 1.1dD (6.00) 0.8eC (5.98) 1.8fC (6.02) 1.5fB (6.01)
Lowered (pH) 88.2 76.7 72.8 59.3 52.7 46.2 36.9
± ± ± ± ± ± ±
0.7aA (6.00) 1.6bA (5.86) 1.9cC (5.74) 2.0dC (5.72) 2.1eC (5.70) 3.5fC (5.69) 3.9gB (5.67)
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a–g : Values without a common letter in superscripts differ significantly (P < 0.05) within the same column. A–D : Values without a common letter in superscripts differ significantly (P < 0.05) within the same row. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C. Table 5 Sperm motility and membrane integrity (% spermatozoa with membrane intact) during liquid storage of egg-yolk coated spermatozoa in mZAP with the initial pH adjusted to 6.04 or 6.25. Storage time (day)
% Progressively motile spermatozoa* pH = 6.04
2 4 6 8 10 12 14 16
85.0 75.9 69.0 59.8 51.6 35.2 18.1 5.2
± ± ± ± ± ± ± ±
0.8A 1.6A 1.5A 1.3A 2.7A 1.7A 0.8A 1.5A
% Spermatozoa with membrane intact
pH = 6.25 89.7 82.9 75.5 73.5 70.1 66.5 55.2 38.6
± ± ± ± ± ± ± ±
1.5A 0.93B 1.3B 1.1B 1.3B 1.3B 1.2B 1.5B
pH = 6.04 92.1 88.7 78.8 70.6 64.6 53.2 37.1 11.4
± ± ± ± ± ± ± ±
0.3A 1.5A 2.0A 2.0A 3.4A 2.8A 4.2A 2.7A
pH = 6.25 93.6 91.2 85.2 84.9 85.2 74.4 70.2 49.9
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. without a common letter in superscripts differ significantly (P < 0.05) within the same row of motility or membrane integrity. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
to those produced with freshly collected semen following AI. 4. Discussion The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually in either mZAP or n-mZAP extenders. Our correlation analysis revealed a strong correlation between sperm motility and semen pH value during the storage. Furthermore, while increasing initial extender pH
Fig. 3. A titration test that compares the buffering capacity between mZAP and n-mZAP extenders. The pH of both mZAP and n-mZAP extenders were first adjusted to 6.5, and then, pH of the extenders was measured as they were titrated with increasing volumes of 5 N HCl.
A,B
± ± ± ± ± ± ± ±
1.9A 2.2A 4.1B 3.2B 2.7B 3.3B 5.0B 3.3B
: Values
from 6.04 to 6.25 or storage with artificially stabilized pH improved sperm quality, storage with gradually lowered extender pH impaired sperm motility. The data suggested metabolic acidosis impaired sperm function during longterm storage of liquid semen. Both mZAP and n-mZAP contained glucose during long-term storage of liquid goat semen. It is accepted that spermatozoa can metabolize glucose through glycolysis for energy supply (Kamp et al., 1996), producing lactate. In the pig, in the presence of glucose, all visible compartments of the spermatozoon as well as the extender were acidified to pH 6.2 within 20 h (Kamp et al., 2003) from the initial pH 7.2 to 7.5 in freshly ejaculated semen (Johnson et al., 2000). Lactate, and several other permeant weak acids, have been shown to reduce the intracellular pH of bovine spermatozoa and many other types of cells (Acott and Carr, 1984). Although it has been reported that depression of intracellular pH by weak acids can reversibly inhibit sperm motility (Jones and Bavister, 2000), which is good for keeping spermatozoa viable during manipulation or preservation (Johnson et al., 2000), it has been suggested that metabolic acidosis may prevent prolonged storage of semen if no proper buffer is present in extenders. The present results supported this speculation by showing that increasing the initial pH of mZAP from 6.04 to 6.25, or storage in n-mZAP with a higher buffering capacity, significantly extended sperm viability during liquid storage of goat semen. One study also showed that sperm motility
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Table 6 Sperm motility, membrane integrity and acrosome intactness during liquid storage of egg-yolk coated spermatozoa in pH 6.25 n-mZAP or mZAP. Storage time (days)
Extenders
pH value
2
n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP n-mZAP mZAP
6.25 6.24 6.15 6.12 6.11 6.03 6.05 5.98 6.01 5.90 5.99 5.88 5.93 5.81
10 12 14 16 18 20
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.04a 0.06a 0.04a 0.07a 0.03b 0.05a 0.02b 0.03a 0.09b 0.04a 0.05b 0.04a 0.03b
% Progressively motile spermatozoa* 88.6 89.1 69.6 64.3 67.4 56.0 57.4 44.7 38.5 33.0 22.0 16.9 11.9 6.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
% Spermatozoa with membrane intact
0.5a 0.5a 0.9a 1.0a 1.0a 1.4b 1.3a 1.8b 0.9a 1.9a 1.2a 2.0b 1.8a 1.4a
91.8 92.9 77.6 74.6 75.3 68.0 66.7 56.9 51.4 43.3 34.1 28.3 23.2 14.3
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3a 0.5a 1.2a 1.7a 1.0a 1.5b 1.1a 1.8b 1.6a 1.3b 1.4a 2.2b 2.4a 2.1b
% Spermatozoa with acrosome intact 90.4 90.9 76.5 75.1 77.3 69.4 67.8 59.9 53.0 46.0 35.9 31.3 23.9 18.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.6a 0.4a 1.1a 1.1a 1.2a 1.3b 1.0a 1.6b 1.4a 1.1b 2.2a 1.3a 2.4a 1.8a
Each treatment was repeated 3 times using ejaculates from three different goats, i.e. each replicate contained one ejaculate from each goat. a,b : In the same column, values with different letters in superscripts differ significantly (P < 0.05) on the same day of semen storage. * These progressively motile sperm data were obtained using Computer-Assisted Semen Analysis (CASA). Storage was at 5 ◦ C.
decreased significantly when the initial extender pH was reduced from 6.04 to 5.54 (Xu et al., 2009). Exposure to mild acidity acidifies the intracellular pH of human spermatozoa and is rapidly spermicidal (Olmsted et al., 2000). One study found that sperm motility, membrane integrity and acrosomal intactness were significantly higher when extender was renewed at 48-h intervals than when it was not renewed during goat semen storage (Zhao et al., 2009). Extender renewal could have improved sperm function by stabilizing pH, removing detrimental metabolites, or by supplying new nutrients. The present results showed that whereas pH value declined with time without extender renewal, it remained constant when semen was stored with regular extender renewal. When extender renewal was conducted using extenders with artificially lowered pH, however, motility was significantly impaired, particularly in non-coated spermatozoa. The results suggested that extender renewal improved sperm quality by removing acidic metabolites and maintaining a stable pH. It is also possible that renewal of the extender eliminated potential damaging entities such as degradation products from degenerating spermatozoa. The present results demonstrated that sperm coating with yolk improved motility by increasing their tolerance to pH decline during liquid semen storage. Yolk is commonly found in semen extenders to preserve spermatozoa against cold shock during the freeze–thaw process. Although the precise mechanism by which yolk protects
spermatozoa is not quite clear, many authors have proposed that the low density proteins (LDL) contained in the yolk could be largely responsible for sperm protection (Evans et al., 1968; Pace and Graham, 1974; Quinn et al., 1980). In fact, some authors have suggested that LDL could adhere to cell membrane during the freeze-thaw process, thus preserving sperm membranes (Foulkes, 1977; Polge, 1980; Graham and Foote, 1987). Furthermore, yolk may also have antioxidant effects during sperm cryopreservation (Alvarez-Rodríguez et al., 2013). Thus, the present results have added a new function to yolk that increases sperm tolerance to pH fluctuations during semen storage. In this study, when semen was stored without extender renewal, the pH value became relatively stable from day 6 of storage onwards; however, sperm motility continued to decline thereafter. Similarly, although pH was maintained constant with extender renewal, sperm motility declined particularly during the later part of the storage. The data suggested that factors other than pH impaired sperm quality particularly during the later part of semen storage. Studies have demonstrated that human spermatozoa are capable of generating reactive oxygen species (ROS), and ROS production in semen has been associated with loss of sperm motility, decreased capacity for sperm-oocyte fusion and loss of fertility (Griveau and Le Lannou, 1997). Lipid peroxidation is significantly accelerated in populations of defective spermatozoa exhibiting high levels of ROS production or in normal cells stimulated to produce oxygen
Table 7 Rates of pregnancy, abortion and kidding after AI using goat semen liquid stored in n-mZAP for different days.* Storage time (days)
% Pregnancy (pregnant/ inseminated does)
% Abortion (aborted/pregnant does recorded)
Average number of kids (kids/does recorded)
0 12 15
79.3 (23/29)a 65.7 (25/38)a 20.0 (5/25)b
16.7 (3/18)a 13.6 (3/22)a 0 (0/2)a
1.93 ± 0.18 (29/15)a 1.78 ± 0.13 (34/19)a 2.00 ± 0.00 (4/2)a
a,b : In the same column, values with different letters in superscripts differ significantly (P < 0.05). Percentage data for pregnancy and abortion rates were analyzed with Chi-squarer (x2 ) test, while data for live kids per pregnant doe were analyzed with ANOVA as described in Section 2. * These pregnancy rates were achieved by adjusting the sperm concentration so that a consistent number of motile spermatozoa were inseminated.
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radicals (Aitken et al., 1989). Furthermore, our recent study observed a remarkable rise in the oxidative stress index (OSI) on day 14 of goat semen storage in n-mZAP (data not shown). In this study, with the same numbers of progressively motile spermatozoa inseminated, pregnancy rates were significantly lower when semen was stored for 15 d than when semen was stored for 0 or 12 d. While sperm motility, membrane integrity and acrosome intactness were similar between day 10 and day 12, they decreased by about 10% between day 12 and day 14 of semen storage in n-mZAP (Table 6). Similarly, although OSI did not differ between day 4 and day 12, it increased significantly on day 14 of goat semen storage in n-mZAP (data not shown). Sperm motility, while an essential feature of healthy spermatozoa, is not necessarily indicative of fertilizing capacity (Hafez, 1992). Normal spermatozoa lose fertilizing ability before they lose motility. In summary, the present results showed that during long-term liquid storage of goat semen, a gradual decline in pH was closely correlated with a decrease in sperm motility. Extender renewal improved sperm motility by maintaining a stable pH, while sperm coating improved motility by increasing their tolerance to pH decline. With increased initial pH and higher buffering capacity, n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than mZAP did. Goat semen liquid stored in n-mZAP for 12 d produced pregnancy and kidding rates similar to those obtained with freshly collected semen following AI with the sperm concentration adjusted so that a consistent number of motile spermatozoa were inseminated. The data are important for protocol optimization for long-term semen storage not only in the goat but also in other species. Conflicts of interest We have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled: Effects of pH during liquid storage of goat semen on sperm viability and fertilizing potential. Acknowledgements This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (No. 31272444) and the Animal Breed Improvement Program of Shandong Province. References Acott, T.S., Carr, D.W., 1984. Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of pH and a quiescence factor. Biol. Reprod. 30, 926–935. Aitken, R.J., Clarkson, J.S., Fishel, S., 1989. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 41, 183–197. Alvarez-Rodríguez, M., Alvarez, M., Anel-López, L., Martínez-Rodríguez, C., Martínez-Pastor, F., Borragan, S., Anel, L., de Paz, P., 2013. The antioxidant effects of soybean lecithin- or low-density
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