The Effect of Whole Ejaculate Filtration on the Morphology and the Fertility of Bovine semen E. F.

GRAHAM

Department of Animal Science University of Minnesota 51. Paul 55108

J. K. GRAHAM Department of Molecular and Celt Biology Pennsylvania State University University Park 16801

ABSTRACT

(Key words: filtration, spermatozoa, fertility)

A Sephadex G-15 filtration method was developed to remove abnonnal and nonmotile bull spenn from an entire ejaculate. The efficiency of filtration was detennined by adding freeze-killed spenn to the ejaculate or using ejaculates with elevated numbers of abnormal cells induced by scrotal insulation. A fertility trial, using split ejaculates, compared fertility of filtered and unfiltered semen. After filtration, samples with 0, 25, and 50% killed cells added contained 77 to 81% motile cells. Addition of 75% killed spenn resulted in significant decrease (52%) of motile cells following filtration. Morphologic examination of semen with elevated numbers of abnormal cells revealed higher percentages of sperm with nonnal shaped heads and normal or swollen acrosomes after filtration than in unfiltered samples. Percentage of pear-shaped heads, lifted acrosomes, and bent tails decreased after filtration. Six high fertility and six low fertility bulls were used to evaluate fertility of filtered semen. Filtering increased motile spenn from 51 to 57% and from 36 to 50% for the high and low fertility bulls, respectively. The 60- to 9O-d nonretum rates for high fertility bulls were not increased by filtering (73 vs. 72%). However, filtering significantly improved the nonreturn rates for the low fertility bulls (61 vs. 67%).

INTRODUCTION

In a successful AI organization, it is important to obtain high fertility (HF) from every semen sample. The negative influence of dead and abnormal spermatozoa on fertility has been reported (13, 20, 22, 28). The first successful separation of motile from nonmotile spermatozoa was performed by passing diluted semen through a layer of small glass beads (2). Other methods of sperm separation have been developed using pyrex beads (17), bovine serum albumin (BSA) gradients (4, 6, 7, 8, 26. 29), glass wool (16), Newtonian gels (15), Sephadex gels (9, 10, 14), and a swimup method (19). Spenn separated on BSA gradients increased motility but failed to raise fertility over that of unseparated semen in the pig (4) and the horse (8). Newtonian gels also failed to enhance fertility in separated bull sperm (15). Glass wool filtration of bull semen raised the 60- to 9O-d nonreturn rate from 71 to 77% (16). Using the swimup technique, Parrish et al. (19) reported that heparin-treated bull sperm had fertilization rates of 71 and 79% for control and separated sperm samples, respectively, when sperm were added to bovine ova in vitro. To date, only small volumes of semen have been separated at one time. The objective of this research was to develop a technique that 1) accommodates the whole ejaculate, 2) establishes the types of spermatozoa removed by the filter, and 3) improves the fertility of ejaculates from low fertility bulls.

Received March 30. 1989. Accepced July 17, 1989. 1990 J Dairy Sci 73:91-97

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MATERIALS AND METHODS

Semen for laboratory studies was obtained from the University of Minnesota dairy herd. Semen for fertility studies was obtained from selected HF and low fertility (LF) sires in Minnesota and Wisconsin. The percentage motile sperm were determined using the Sephadex G-15 filtration assay as described by Graham et al. (10). A TEST-york buffer or sodium citrate buffer was used in the laboratory experiments. The sodium citrate buffer contained sodium citrate adjusted to 300 mOsm/kg and a pH of 7.0. The stock solution of TEST was prepared with 48.3 g TES(N-Tris(hydroxymethyl) methyl-2-amino ethane sulfonic acid), 11.6 g TRIS (Tris-(hydroxymethyl)amino methane), and 2 g glucose/ L double deionized water, at 325 mOsm!kg and pH of 7.0. Egg yolk (20% vol/vol) was added and the solution centrifuged at 12,000 x g for 15 min. The supernatant fluid was decanted for use. Glycerol (6% vol/vol), fructose (.2 g/loo ml), penicillin (1000 units/ml), and streptomycin (100 j.lg/ml) were added. Semen was diluted in TEST-yolk extender containing glycerol at 370C to avoid cold shock and cooled to SOC in 2 h. Semen to be frozen was equilibrated at SOC for an additional 2 h prior to freezing. Samples were filtered at 5°C prior to the end of the equilibration time. Semen for freezing was packaged in .S-ml French straws and frozen 4.2 cm above liquid nitrogen for 10 min. The straws were then plunged into liquid nitrogen and stored until used. The filter was prepared by drilling a hole (diameter 1.59 mm) at the 30-ml mark of a 6O-ml plastic disposable syringe. This hole allowed air bubbles in the barrel to escape when the plunger was lowered. A small amount of glass wool was placed in the bottom of the barrel to prevent loss of the Sephadex. Two to three milliliters of the extender were added to wet the glass wool. The plunger was then depressed to remove the extender and compress the glass wool in the barrel bottom. Approximately 50 cm of Tygon tubing (Ld. 3.2 mm) were attached to the tip of the syringe and clamped shut. The tubing was inserted into a filtrate collecting vessel. Dry Sephadex G-IS (3 g) was distributed evenly over the glass wool, and 15 rol extender were added. The Sephadex Joumal of Dairy Science Vol. 73. No. I, 1990

was allowed to swell .5 to 1 h. The extender was removed by releasing the tubing clamp, covering the hole in the barrel with a finger, and manually forcing the plunger down. The plunger was not allowed to pass the hole in the barrel. This prevents the suction created while removing the plunger from lifting the Sephadex-glass wool plug from the bottom of the barrel. The syringe was then secured in a vertical position using a buret clamp and ring stand. Semen that had been extended and cooled to 5°C was gently layered over the Sephadex. Additional extender was placed over the semen to fill the barrel to 30 ml. The plunger was placed in the barrel and lowered to the 3D-ml mark. The filtration column was then attached to a dual infusion and withdrawal pump mounted in a vertical position. Semen was filtered through the prepared filter at a rate of 2.7 ml/min. Extended ejaculated semen from one bull was filtered through each Sephadex-glass wool column. The Sephadex and glass wool were then discarded, the syringe was washed, dried, and fresh glass wool and Sephadex were used for each subsequent filtration. The cost per filtered ejaculate is dependent only upon the cost for glass wool, which is negligible, and for 3 g Sephadex G-IS. The percentage of motile sperm and sperm morphology data were analyzed by analysis of variance. Means were tested using Duncan's new multiple range test (23). Fertility data were analyzed by the chi-square procedure (23). Experiment 1

This experiment evaluated the efficiency of the Sephadex 0-15 filtration system in removing dead sperm from a semen sample. Pooled semen from 2 to 4 bulls was extended with 20 ml TEST-yolk extender, split into treatment groups, and cooled to 5°C. Different volumes of freeze-killed sperm were then added (Table 1). A volume of .5 ml was removed from each treatment to assess the percentage of motile sperm. An additional .5-ml volume was removed, diluted with 3 ml TEST-yolk, and prepared for cryopreservation as the unfiltered control. The remainder of the sample was filtered and frozen as previously described. This study was repeated with five pooled samples (replicates).

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FILTRATION OF BOVINE SPERMATOZOA

TABLE 1. The volwne of raw semen. freeze-killed semen. and TEST1-yolk extender added for ratios of live to dead spermatozoa for filtration. Percentage freeze-killed semen

Constituent Raw semen, mI Freeze-killed semen. mI TEST-Yolk. ml Total volwne, m1

o 3

o 12

15

25

50

75

3 3 3 1 3 9 11 15

9 15

3 15

100

o 5 10

15

lTEST:TES 48.3 g. 11.6 g Tris, 2 g glucoseIL water at pH 7.0. 325 mOsm/kg.

Experiment 2

The efficiency of the filtration procedure in removing abnormal cells from an ejaculate was determined in Experiment 2. Modifications of Gustafsson's (11) procedure were used to insulate the scrotum of two bulls. A nylon bag filled with polyester fibers was placed around the scrotum. The bag was held in place by adhesive tape wrapped anterior to the testes. The bag was placed over the scrotum for a 24-h period and removed for the following 24 h. This procedure was repeated five times. The temperature inside the bag was measured using a II-em rectal thermometer (35 to 36.5'C). Scrotal skin temperature was determined on the posterior aspect of the scrotum of the right testicle (34.5 to 35.5'C). Two ejaculates from each of the two bulls were collected twice weekly. Ejaculates from 7 collection d were selected for filtration based on the presence of the maximum numbers of abnormal spermatozoa, which occurred during wk 3 to S after initiation of scrotal insulation. Spermatozoa for morphological studies were fixed in formalin-buffered saline (S) immediately after treatment and stored at S'C until analyzed. Three replicates from each ejaculate were sampled. Each sample was examined five times; each time 200 ceUs were observed for a different morphological feature. The abnormalities examined using phase contrast microscopy (1000>

The effect of whole ejaculate filtration on the morphology and the fertility of bovine semen.

A Sephadex G-15 filtration method was developed to remove abnormal and nonmotile bull sperm from an entire ejaculate. The efficiency of filtration was...
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