R.M. Kenney

The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion

The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percentage pregnant per first cycle (FCP; SD alpha t, r = -0.30, P < or = 0.01; % COMP alpha t, r = -0.42, P < or = 0.0001); and the percentage pregnant per cycle (PC; mean alpha t, r = -0.31, P < or = 0.01; SD alpha t, r = -0.32, P < or = 0.01; % COMP alpha t, r = -0.41, P < or = 0.0001). This study describes detectable intrinsic variation in sperm chromatin structure among fertile stallions (SPR, mean = 83%; FCP, mean = 58%; PC, mean = 57%) in an active breeding population (number of mares bred/stallion/year, mean = 37), in the absence of overt reproductive abnormalities and apparent diseases such that an increase in the susceptibility of sperm DNA to denaturation is associated with reduced fertility, both in terms of efficiency of reproduction (FCP and PC) and seasonal pregnancy rate (SPR). Both COMP alpha t and mean alpha t were useful indicators of fertility, with COMP alpha t being the only SCSA value able to identify mean differences between fertility groupings for SPR and FCP, and overall it was the most reliable indicator of fertility in this group of stallions. The SCSA is able to evaluate a compartment of the spermatozoa which is different from that of traditional tests for sperm quality such as motility and morphology.

Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fractions was combined with 50% of the original volume of SP fractions for each ejaculate to represent total ejaculates. SR and total ejaculates were diluted with skim milk-glucose semen extender as follows: 1) no dilution, or dilution to 2) 100 x 10(6)sperm/ml, 3) 50 x 10(6)sperm/ml, or 4) 25 x 10(6)sperm/ml. Semen samples were evaluated at 0.5, 3, 6, 12, and 24 h postejaculation (25 degrees C storage temperature) for percentages of total spermatozoal motility (TSM) and progressive spermatozoal motility (PSM). Mean TSM was greater (P < 0.05) in SR ejaculates than total ejaculates at 12 and 24 h postejaculation. Mean TSM of undiluted semen was lower (P < 0.05) than other dilution ratios over all periods. Mean TSM was greater (P < 0.05) at a 25 x 10(6)sperm/ml dilution ratio than a 50 x 10(6)sperm/ml dilution ratio at 12 and 24 h postejaculation, and greater (P < 0.05) than a 100 x 10(6)sperm/ml dilution ratio from 3 to 24 h postejaculation. Similar patterns were found for PSM. Collection of SR ejaculates and dilution to 25 x 10(6)sperm/ml improved longevity of spermatozoal motility.

Use of altrenogest to prepare ovariectomized mares as embryo transfer recipients.

Altrenogest was administered to ovariectomized mares to determine if treatment would enable establishment and maintenance of pregnancy after transfer of a 7-d embryo. Three different treatment regimens were used: Group A received 22 mg altrenogest daily starting 5 d before transfer, Group B received 66 mg altrenogest daily starting 6 days before transfer, Group C received 300 mg progesterone in oil intramuscularly daily starting 5 d before transfer. Intact, ovulation-synchronized recipients were used as controls for transfer technique. Pregnancy rates were 1 6 , 2 6 , 2 5 , and 13 19 for Group A, Group B, Group C, and controls, respectively. The pregnancy rate in Group A was significantly different from controls and Group A mares had poor uterine and cervical tone. These results show that ovariectomized mares treated with altrenogest are capable of establishing pregnancy after embryo transfer. Treatment with 22 mg altrenogest appears to be insufficient for optimal pregnancy rates after transfer in ovariectomized recipients.

Use of a semen extender containing antibiotic to improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa.

A breeding trial was conducted to determine if a semen extender containing polymixin-B sulfate would improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa . Twenty-three mares were bred to the stallion by one of three methods: artificial insemination with raw semen (Group 1, n = 10), artificial insemination with semen mixed 1:1 with a nonfat dry skim milk/glucose extender containing 1000 units/ml polymixin-B sulfate (Group 2, n = 9), or natural service immediately following infusion of the uterus with 100 ml of the same extender (Group 3, n = 4). Artificial breedings contained a minimum insemination dose of 500 x 10(6) progressively motile spermatozoa. All mares were bred every other day while in estrus. Pregnancy status was determined by transrectal ultrasound examination 15 d after the last breeding. First-cycle pregnancy rate for Group 2 mares (78%) was greater (P < 0.01) than for Group 1 mares (10%). There was a tendency (P = 0.10) for the pregnancy rate of Group 3 mares (50%) to be greater than Group 1 mares. The use of a semen extender containing polymixin-B sulfate improved the fertility of this stallion.

Aspiration of oocytes from mature and immature preovulatory follicles in the mare.

Two experiments were conducted to investigate methods for aspirating oocytes from immature preovulatory follicles in the mare. In Experiment 1, the ovary was manipulated per rectum and the follicle was punctured by a needle introduced through the flank. Suction was provided by either a syringe or by a vacuum pump connected to the needle via tubing. The preovulatory follicle was aspirated when it reached a diameter of 32 +/- 2 mm (Group A); 37 +/- 2 mm (Group B); or 42 +/- 2 mm (Group C). There was no significant difference in oocyte recovery rates between the two methods (7/24 vs 3/19). Oocyte recovery rates were higher for Groups B and C (5/14 and 4/12, respectively) than for Group A (1/17; P < 0.05). In Experiment 2, the ovary was held against the internal abdominal wall by the hand inserted into the abdomen via a vaginal incision, and the follicle was flushed after aspiration. Recovery rates were 9/13 (69%) for mares treated with human chorionic gonadotrophin (hCG) and 15/21 (71%) for unstimulated mares. This difference was not significant. The oocyte recovery rate for unstimulated follicles (average diameter 39.7 mm) in Experiment 2 was significantly higher than those for Group B and Group C in Experiment 1 (P < 0.05).

Conditioned Suppression of Sexual Behavior in Stallions and Reversal With Diazepam

Sexual behavior dysfunction unaccompanied by detectable physical or endocrine abnormality is an important cause of reproductive failure among domestic stallions. Several authors have suggested that such dysfunction may be psychogenic, related to negative experience associated with intense handling and training. An experimental model of experience-related dysfunction was developed by exposing pony stallions to erection-contingent aversive conditioning. This resulted in rapid, specific suppression of sexual arousal and response similar to spontaneously occurring dysfunction. Subsequently, treatment with a CNS-active benzodiazepine derivative (diazepam) reversed these effects.

Comparison of two techniques for obtaining endometrial bacteriologic cultures in the mare

The endometria of 39 mares were cultured simultaneously using a swab guarded with a double cannula and distal, teflon plug and an unguarded swab with a single, open cannula. Sheep blood (5%) agar, Mac-Conkeys agar, and Sabourads agar media were innoculated with each swab. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. There were significantly more plates that failed to yield bacterial or fungal growth when streaked with swab specimens obtained with the guarded cannula than when streaked with those obtained with the unguarded cannula. It was concluded that while culturing the endometrium of mares for bacteria or fungi, the use of a guarded instrument consisting of a double cannula with a closed end will result in the recovery of fewer contaminants; therefore, it will be more likely to result in a more accurate representation of uterine bacterial and fungal flora.

Gonadotropin Releasing Hormone (GnRH) Affects Precopulatory Behavior in Testosterone- Treated Geldings

Twelve pony geldings with (n = 6) and without (n = 6) testosterone replacement (200 micrograms/kg testosterone propionate in oil, SC every 48 hours) received either gonadotropin releasing hormone (GnRH; 25 micrograms SC every 3 hours) or control treatment. Sexual behavior was recorded during 4-minute exposure to an estrous mare, 3 times weekly for 2 weeks before treatment, 3 weeks during treatment, and 3 weeks after treatment had been discontinued. The group receiving testosterone and GnRH (n = 3) exhibited significantly greater flehmen response frequency and attention duration and significantly lower vocalization frequency and erection duration than the group receiving only testosterone (n = 3). GnRH had no apparent effect on sexual behavior in geldings without testosterone replacement (n = 3). These results support an hypothesis of testosterone-dependent, CNS-mediated effects of GnRH on precopulatory behavior in the stallion.

Unilateral hydrosalpinx and absence of the infundibulum in a mare

Both ovaries and associated oviducts were removed from a mare via colpotomy. The right oviduct was greatly enlarged and filled with fluid. No infundibulum could be identified; rather, the blind distended end of the oviduct adhered to the ovary at the ovulation fossa. Two cysts were attached to the exterior of the oviduct a few centimeters from the fossa. Histologic examination of the oviduct and cysts revealed a dilated lumen, a decreased infolding of the wall into the lumen, and a lack of a defined muscle layer. Congenital absence of the infundibulum resulting in a blind ampullar terminus is proposed as the cause of the hydrosalpinx.

Comparison of pregnancy rates achieved with frozen semen using two packaging methods

The pregnancy rate achieved with frozen semen using two packaging methods was compared in two separate experiments. The sperm-rich fraction of the semen was collected and immediately extended in an egg-yolk based extender to a final concentration of 100 x 10(6) sperm/ml. Half of each ejaculate was packaged in 4.0-ml plastic straws and the other half in 10-to 12-ml flattened aluminum tubes. After detection of a 35-mm follicle, mares were inseminated daily until ovulation. Pregnancy was detected, and embryonic vesicle size was measured by ultrasonic echography Days 14 to 26 after ovulation. The one-cycle pregnancy rate achieved using both packaging methods was 50% overall (23 46 ), with 46% (12 26 ) and 55% (11 20 ) rates achieved, respectively, using straws and aluminum tubes. These rates were not significantly different.