Review: Principles of maximizing bull semen production at genetic centers.

Knowledge of the capabilities and limitations of the reproductive capacity of bulls is vital in maximizing reproductive efficiencies. Bull semen collection guidelines established by researchers and industry personnel to maximize the sperm harvest from bulls have been evolving for more than 60 years. Today, a mature artificial insemination industry employs those strategies to meet demands. These efficient management schemes exploit the reproductive potential of each sire while minimizing the associated risk of injury to bulls and reduce associated production costs. Personnel employed by a semen producing facility must be authorized to make effective and rational decisions based on principles of bull sexual behavior and reproductive physiology. Furthermore, collection facilities must be well planned to allow for the safe presentation of novel sexual situations while affording maximum safety for employees and proper footing for bulls. Normal bulls produce and ejaculate tremendous numbers of sperm. Most bulls have a sufficient libido for routine sexual activity, but become satiated to predictable stimulus situations. Frequent changes to the novelty should allow weekly harvest of four to six ejaculates per week for most bulls. Utilizing the physiological characteristics associated with each ejaculate to establish the collection frequency of each bull, and empowering an integrated collection and laboratory staff to monitor and make adjustments to the ejaculation frequency are necessary in maximizing the sperm harvest. Young bulls can ejaculate 10 to 20 billion sperm per week, and mature bulls should ejaculate 40 to 60 billion sperm per week. Semen collection management procedures should be reviewed when bulls do not meet production goals.

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm.

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO(2) in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10(6) sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.

Pregnancy rates in heifers and cows with cryopreserved sexed sperm: effects of sperm numbers per inseminate, sorting pressure and sperm storage before sorting.

Field trials were conducted to increase fertility with AI of flow-sorted, sexed bovine sperm. In the first trial, a novel competitive fertilization approach was used to compare pressures (30psi vs 50psi) for sorting sperm. Both X- and Y-sperm were sorted to approximately 95% purity at 30 and at 50psi; X-50+Y-30 (and the converse) were mixed in equal numbers for AI of heifers. Fetal sex divulged which treatment produced the pregnancy; 82% of pregnancies resulted from the 30psi treatment (P<0.05). Based on a similar approach, a new-pulsed laser did not damage sperm any more than the previous standard continuous wave laser. In a large field trial, sorting sperm at 40psi increased pregnancy rates in heifers relative to 50psi (42.3% vs 34.1%, n=367/group, P<0.05). Storing sperm for 20h before sorting at 40psi decreased pregnancy rates from 42.3% (n=367) to 36.8% (n=368; P<0.05). Breeding heifers with sexed sperm 55-56h after CIDR removal and PGF(2alpha) resulted in 34% (n=32) pregnant, compared to 49% (n=35) with fixed-time insemination 67-68h after CIDR removal (P>0.1). Lactating dairy cows pre-screened for normal reproductive tracts when OvSynch injections (GnRH, prostaglandin, GnRH) were initiated, had similar (P>0.1) pregnancy rates to timed AI, with 10x10(6) sexed sperm (43.9%, n=57), 2x10(6) sexed sperm (40.5%, n=57) and 10x10(6) unsexed control sperm (55.6%, n=58). A final field trial with unselected, lactating dairy cows resulted in similar pregnancy rates for 2x10(6) sexed sperm in 0.25mL straws (25.0%, n=708) and 0.5mL straws (24.4%, n=776), but lower (P<0.05) than unsexed control sperm (37.7%, n=713). Younger cows and those >84 days in milk had the highest pregnancy rates for both sexed and unsexed sperm. These studies improved sperm sexing procedures, and provided insight into appropriate commercial use of sexed sperm.

Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition.

In six field trials, doses between 1.0 and 6.0 x 10(6) total sexed, frozen-thawed sperm were inseminated into the uterine body or bilaterally into the uterine horns of heifers and nursing Angus cows 12 or 24h after observed estrus. Except for one comparison in one trial in which uterine body insemination was slightly superior (P<0.05) to uterine horn insemination, there was no significant (P>0.1) difference between sites of semen deposition. Additionally, except for one small study with limited numbers, there was essentially no difference in pregnancy rates in the range between 1.5 and 6 x 10(6) sexed, frozen-thawed sperm per inseminate. Pregnancy rates with smaller doses of sexed sperm averaged about 75% of controls of 20 x 10(6) total frozen-thawed, unsexed sperm. While 1.0 x 10(6) sexed, frozen-thawed sperm per insemination dose resulted in decreased pregnancy rates compared to larger doses, the lesser fertility with sexed sperm could not be compensated by increasing sperm numbers in the range of 1.5-6 x 10(6) sperm per dose. Pregnancy rates with 2 x 10(6) sexed, frozen-thawed sperm per dose were not markedly less than control pregnancy rates with 20 x 10(6) frozen-thawed unsexed sperm/dose in well-managed herds.

Pregnancy rates in cattle with cryopreserved sexed spermatozoa: effects of laser intensity, staining conditions and catalase.

The overall aim of this research was to improve fertility of cattle inseminated with sexed spermatozoa by improving sperm sorting procedures. Six field trials were conducted in which 4,264 heifers were inseminated into the uterine body with cryopreserved sexed or unsexed control spermatozoa. Pregnancy or calving rates with doses of 2 x 10(6) sexed spermatozoa ranged from 32 to 51%; these averaged 69% of the pregnancy rates with 20 x 10(6) unsexed, control spermatozoa (range 53 to 79% of controls). Fertility of sexed spermatozoa was especially low on farms where control fertility was low. Accuracy of sexing ranged from 86 to 91%. Laser power of 150 mW for interrogating spermatozoa did not result in lower pregnancy rates (43%) than when power was decreased as much as possible for a particular sorting batch (50 to 130 mW) to still achieve sexing accuracy (38% pregnant). Addition of catalase to fluids containing spermatozoa was beneficial when thawed spermatozoa were incubated in vitro for 2 h but had no effect on pregnancy rates. There also was no effect on pregnancy rates between two concentrations of Hoechst 33342 for staining spermatozoa. Freezing 2 x 10(6) sexed spermatozoa at 20 x 10(6)/ml resulted in a slightly higher rate of pregnancy (P < 0.05) than at 10 x 10(6)/ml. The information obtained in these trials, along with other improvements, notably lowering pressure in the sorting system from 50 to 40 psi, has been used to improve procedures for sexing spermatozoa commercially.

Embryo production from superovulated cattle following insemination of sexed sperm.

Two trials were conducted to ascertain fertilization rate, embryo quality and numbers of transferable embryos in superovulated heifers and cows inseminated with sexed sperm. Inseminates contained 2 x 10(6), 10 x 10(6) or 20 x 10(6) total sperm enriched for the X- or Y-chromosome ( approximately 90%) by flow cytometry/cell sorting. Non-sexed inseminates contained 40 x 10(6) total sperm. Donors in each trial were allocated to one of each of the bulls included in that study. Each donor was inseminated with frozen/thawed sperm from the same bull for each treatment in successive courses of superstimulation with twice daily i.m. injections of FSH for 4 d. Heifers and cows were inseminated 12 and 24 h after visually observed standing estrus in Trial 1. In Trial 2, a single timed inseminate was used 70-72 h following PGF(2alpha). Ova/embryos were collected non-surgically 7-7.5 d after insemination. In both trials, fewer ova were fertilized with sexed versus non-sexed treatments and with 2 x 10(6) sexed sperm compared to higher doses (P < 0.05). However, insemination of 20 x 10(6) total sexed sperm of >or=90% purity resulted in similar numbers of transferable embryos of the desired sex compared to that for non-sexed sperm.

High pressure flow cytometric sorting damages sperm.

Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 microm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 50 psi with the 70 microm nozzle tip increased sperm motility post-thaw at 30 min and 2h from 40.5 to 48.0% and 30.0 to 40.2%, respectively (P<0.05). In Experiment 2, 49, 43, 37, 31, and 25 psi resulted in 24.2, 32.8, 35.6, 37.5, and 39.8% progressively motile spermatozoa post-thaw (P<0.05). In Experiment 3, 3 pressures (50, 40, 30 psi)x2 sorting methods were further evaluated. At 50, 40, and 30 psi, respective mean sperm motilities at 30 min were 44.8, 48.6, and 49.6% (P<0.05), and percentage of live spermatozoa were 51.7, 55.7, and 57.8% (P<0.05). The improvement of post-sort sperm quality with lowered pressure was also evident in stallion spermatozoa. After sorting at 30, 40 and 50 psi were 40.6, 34.5 and 30.1% motile spermatozoa (P<0.1), and were 76.7, 72.5 and 67.8% (P<0.05) live spermatozoa (determined by SYBR-14/propidium iodide staining). In Experiment 4 sorter performance was evaluated with two pressures (40 and 50 psi)x2 staining concentrations of bovine spermatozoa (75 x 10(6) and 100 x 10(6)mL(-1)). Lowering pressure to 40 psi did not lower sort rate and purity when compared to 50 psi (P>0.05), and higher sperm concentration during staining increased sort rate (P<0.05). In conclusion, lowering pressure of the MoFlo SX flow cytometer for sperm sorting from 50 psi (standard pressure) to 40 psi clearly improved sperm quality without a significant decrease in sorter performance.

Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa.

The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.

Hysteroscopic insemination of mares with low numbers of nonsorted or flow sorted spermatozoa.

The objectives of this study were 1) to compare pregnancy rates resulting from 2 methods of insemination using low sperm numbers and 2) to compare pregnancy rates resulting from hysteroscopic insemination of 5 x 106 nonsorted and 5 x 106 spermatozoa sorted for X- and Y-chromosome-bearing populations (flow sorted). Semen was collected with an artificial vagina from 2 stallions of known acceptable fertility. Oestrus was synchronised (June to July) in 40 mares, age 3-10 years, by administering 10 ml altrenogest orally for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. All mares were given 3000 iu hCG i.v. at the time of insemination to induce ovulation. Mares were assigned randomly to 1 of 3 treatment groups: mares in Treatment 1 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited deep into the uterine horn with the aid of ultrasonography. Mares in Treatment 2 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited onto the uterotubal junction papilla via hysteroscopic insemination. Mares in Treatment 3 (n = 20) were inseminated using the hysteroscopic technique with 5 x 10(6) flow sorted spermatozoa. Spermatozoa were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Pregnancy was determined ultrasonographically at 16 days postovulation. Hysteroscopic insemination resulted in more pregnancies (5/10 = 50%) than did the ultrasound-guided technique (0/10 = 0%; P<0.05) when nonsorted sperm were inseminated. Pregnancy rates were not significantly lower (P>0.05) when hysteroscopic insemination was used for sorted (5/20 = 25%) and nonsorted spermatozoa (5/10 = 50%). Therefore, hysteroscopic insemination of low numbers of flow sorted stallion spermatozoa resulted in reasonable pregnancy rates.

Insemination of mares with low numbers of either unsexed or sexed spermatozoa.

Two experiments were conducted to determine pregnancy rates in mares inseminated 1) with 5, 25 and 500 x 10(6) progressively motile spermatozoa (pms), or 2) with 25 x 10(6) sex-sorted cells. In Experiment 1, mares were assigned to 1 of 3 treatments: Group 1 (n=20) was inseminated into the uterine body with 500 x 10(6) pms. Group 2 (n=21) and Group 3 (n=20) were inseminated into the tip of the uterine horn ipsilateral to the preovulatory follicle with 25 and 5 x 10(6) pms, respectively. Mares in all 3 groups were inseminated either 40 (n=32) or 34 h (n=29) after GnRH administration. More mares became pregnant when inseminated with 500 x 10(6) (18/20 = 90%) than with 25 x 10(6) pms (12/21 = 57%; P<0.05), but pregnancy rates were similar for mares inseminated with 25 x 10(6) vs 5 x 10(6) pms (7/20 = 35%) (P>0.1). In Experiment 2, mares were assigned to 1 of 2 treatments: Group A (n=11) was inseminated with 25 x 10(6) spermatozoa sorted into X and Y chromosome-bearing populations in a skimmilk extender. Group B (n=10) mares were inseminated similarly except that spermatozoa were sorted into the skimmilk extender + 4% egg yolk. Inseminations were performed 34 h after GnRH administration. Freshly collected semen was incubated in 224 microM Hoechst 33342 at 400 x 10(6) sperm/mL in HBGM-3 for 1 hr at 35 degrees C and then diluted to 100 x 10(6) sperm/mL for sorting. Sperm were sorted by sex using flow cytometer/cell sorters. Spermatozoa were collected at approximately 900 cells/sec into either the extender alone (Group A) or extender + 4% egg yolk (Group B), centrifuged and suspended to 25 x 10 sperm/mL and immediately inseminated. Pregnancy rates were similar (P>0.1) between the sperm treatments (extender alone = 13/10, 30% vs 4% EY + extender = 5/10, 50%). Based on ultrasonography, fetal sex at 60 to 70 d correlated perfectly with the sex of the sperm inseminated, demonstrating that foals of predetermined sex can be obtained following nonsurgical insemination with sexed spermatozoa.

Effects of follicular fluid or progesterone on in vitro maturation of equine oocytes before intracytoplasmic sperm injection with non-sorted and sex-sorted spermatozoa.

In Expt 1, compact cumulus oocyte complexes (COCs) were matured in: (i) control medium (Hepes-buffered TCM-199 with 10% oestrous cow serum (OCS) + oestradiol, LH and FSH); (ii) Hepes-buffered TCM-199 with 20% follicular fluid; or (iii) control medium containing 250 ng progesterone ml(-1). Mature oocytes were collected by transvaginal aspiration as a positive control for the in vitro maturation (IVM) treatments. Oocytes were fertilized by ICSI and cultured in Menezo's B2 + 5% fetal calf serum (FCS). There were no significant differences among IVM treatments. In Expt 2, oocytes with expanded COCs were matured in Hepes-buffered TCM-199 with 10% OCS, oestradiol, LH and FSH with different concentrations of progesterone (0, 50, 250 and 1250 ng ml(-1)). Oocytes were fertilized by ICSI and cultured in a chemically defined medium. The medium containing 1250 ng progesterone ml(-1) resulted in fewer oocytes with a visible first polar body after maturation (P < 0.05), whereas the media containing 0 and 50 ng progesterone ml(-1) resulted in higher development rates to seven- to eight-cell embryos (P < 0.05), compared with media containing 250 or 1250 ng progesterone ml(-1). Six of the resulting morulae were transferred to recipient mares. In addition, oocytes (n=32) from Expt 2 were injected with sex-sorted spermatozoa, obtained by separating X- and Y-bearing spermatozoa with a Cytomation MoFlo flow cytometer/cell sorter. Two embryos resulting from ICSI with X-bearing spermatozoa were transferred to the oviduct of a recipient mare. No pregnancies were established after transfer of embryos in these experiments.

Cryopreservation of flow-sorted bovine spermatozoa.

Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg-yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years.

Insemination of heifers with sexed sperm.

Data from inseminating 1,000 heifers consecutively with sexed sperm and 370 heifers with control sperm in 11 small field trials are summarized. Semen was from 22 bulls of unknown fertility of various beef and dairy breeds, and 6 inseminators participated. Freshly collected sperm were sexed using a MoFlo flow cytometer/cell sorter after staining sperm with the DNA-binding dye Hoechst 33342; the principle is that the bovine X chromosome has 3.8% more DNA than the Y chromosome. Accuracy approaching 90% males or females was achieved. There was little difference in pregnancy rates between sexed, unfrozen and sexed, frozen sperm. In 5 of 6 field trials, there was little difference in pregnancy rates between insemination doses of 1.0 to 1.5 x 10(6) versus 3.0 x 10(6) sexed, frozen sperm. In the most recent trials, pregnancy rates with sexed, frozen sperm were within 90% of unsexed, frozen controls that had 7 to 20 times more sperm/insemination dose; however, in a few trials, control pregnancy rates were substantially higher than with low doses of sexed sperm. There were too few inseminations per bull to test bull differences in pregnancy rates rigorously. Insemination of sexed, frozen sperm bilaterally into the uterine horns produced pregnancy rates similar to insemination into the uterine body in 4 of 5 field trials. Pregnancy rates among inseminators did not differ significantly. There was no excess embryonic death between 1 and 2 months of gestation with pregnancies from sexed sperm, and very few abortions occurred between 2 months of gestation and term. Although rigorous epidemiological studies remain to be done, calves resulting from sexed sperm appear to exhibit no more abnormalities than controls.

Effects of extender and insemination dose on postthaw quality and fertility of bovine sperm.

Quality and fertility of sperm extended in egg yolk-Tes-Tris were compared with those of sperm in egg yolk-citrate and homogenized milk extenders. Extended semen was frozen in .5-ml plastic straws at 11, 15, 17, or 22 X 10(6) sperm per insemination dose. Laboratory evaluations at 0, 1, 2, and 4 h after thawing semen utilized four tests of spermatozoal quality. Use of egg-yolk citrate extender resulted in a higher percentage of progressively motile sperm as determined visually at 0 h after thawing than use of egg yolk-Tes-Tris or homogenized milk extenders. Sperm extended in egg yolk-citrate had 18% lower activity of bound amidase at 0 h than sperm extended in egg yolk-Tes-Tris. The 75-d nonreturn rates were affected by insemination dose but not be extender or the interaction of extender and insemination dose. Fertility was lower after insemination of 11 X 10(6) sperm than for pooled data for the three higher insemination doses (64 vs. 68%). Based on all data, postthaw quality of sperm processed in the one-step egg yolk-Tes-Tris extender was similar to that for sperm extended in egg yolk-citrate or homogenized milk.

Effects of exposing bovine sperm to bovine serum albumin, or freeze-thawing, on sperm-bound amidase activity.

Isolation of a self-selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm-bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm-bound amidase (P less than .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5 degrees C or layered onto a solution of 6% BSA in extender at 37 degrees C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5 degrees C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm-bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P less than .01). However, the amount of exposed sperm-bound amidase also was greater (P less than .05); this was a deleterious change. Immediately after thawing, more (P less than .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37 degrees C there was no difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm-bound amidase activity as a marker for acrosomal integrity in bull spermatozoa.

An assay for sperm-bound amidase activity was validated using bovine spermatozoa and N-benzoyl-DL-arginine p-nitroanilide as substrate. The assay had intra- and interassay coefficients of variations of 5 and 12%, respectively. It is an inexpensive, simple and rapid assay since 100 samples can be evaluated in 2 hours and it requires only 4 X 10(6) spermatozoa per sample. Sperm-bound amidase activity was proportional (r = 0.95) to the percentage of spermatozoa with an intact acrosome, as determined by differential interference-contrast microscopy. A change of five percentage units in the incidence of damaged spermatozoa was detectable. Using this procedure, assessment of sperm-bound amidase activity is therefore a sensitive and efficient means of evaluating acrosomal integrity.