Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm.

An acrosomal staining technique that can differentiate between living and dead sperm was developed for equine sperm. The fluoresceinated lectin Pisum sativum agglutinin (FITC-PSA) was used to identify the presence or absence of acrosomal contents, while the supravital nuclear dye Hoechst 33258 (H258) was used to assess viability. The accuracy of the FITC-PSA acrosomal stain was tested by comparing the percentage of sperm that had lost their acrosomal contents, detected by the staining method, with that detected by transmission electron microscopy (TEM). Following capacitation in vitro, the acrosomal status of sperm induced to acrosome react with A23187 and of control sperm were very similar with the staining technique and TEM, confirming the accuracy of the FITC-PSA acrosomal stain. We investigated the relationship between viability as measured by exclusion of H258 and motility as measured by three methods: one subjective and two objective. Although there was a good correlation between viability and motility as measured by all three methods (r = 0.88, 0.85, 0.75), there was always a proportion of viable sperm that were nonmotile. The physiology of the viable, nonmotile sperm was further investigated by comparing for individual sperm the viability as measured by exclusion of H258 with the mitochondrial function as measured by rhodamine 123. A good correlation (r = 0.99) was found to exist between viability and mitochondrial function, indicating that viable, nonmotile sperm possess functional mitochondria and confirming the ability of supravital staining to distinguish between living and dead sperm. We determined that 29-81% of the sperm in semen that had lost their acrosomal contents were in fact dead. Thus, this acrosomal staining technique can provide more relevant endpoints for future investigations of capacitation, the acrosome reaction, and sperm handling techniques in the horse.

Plasma prolactin concentrations in mares and their neonates after oxytocin induction of parturition.

Studies were undertaken to investigate the effects of oxytocin induction on prolactin release in term (Group II) and preterm (Group III) mares and to compare these effects to spontaneously foaling mares (Group I). Since physiological concentrations of prolactin in blood have not been measured in the neonatal foal, experiments were designed to monitor prolactin in the cord artery and jugular blood of the foals from all groups of mares. Although prolactin levels varied in term mares (Group I and II) during the last 11 days of pregnancy, an increase was observed between Day -6 and Day 0 (2.7 and 11.9 ng/ml respectively; P less than 0.1). The average concentration of prolactin over the last 4 days (Days -3 to 0) had increased by 40% when compared to the average concentration on Days -6, -5, and -4. These findings indicate a rising trend which appears to occur concomitantly with changes in concentrations of 2 mammary components tested, sodium and potassium. Prolactin concentrations did not significantly increase in term mares after oxytocin treatment or in spontaneously foaling mares. However, the preterm induced mares had higher prolactin concentrations during the first stage of labor (19.3 +/- 7.2 ng/ml) than prior to treatment with oxytocin (4.7 +/- 2.0 ng/ml; P less than 0.01). Levels of prolactin in all groups significantly declined by 20-min post-placental expulsion. For the first 30 min after birth, prolactin concentrations in foals from oxytocin-induced mares appeared to be 2-fold higher than those from spontaneously foaling mares. Thereafter, prolactin values declined to baseline values by 48 hrs. When comparing cord arterial plasma with cord venous plasma in each group, prolactin concentrations were similar. However, the average prolactin levels in both the cord artery and vein appeared higher (ave: 1.1 ng/ml) in Group II and III than in Group I (less than 0.5 ng/ml). From these results, the authors suggest that 1) prolactin may have a role in regulating mammary secretory products in mares just prior to parturition; 2) oxytocin may increase prolactin secretion in preterm induced mares; 3) oxytocin induction may have a short term effect to increase circulatory prolactin concentrations in neonates in utero regardless whether their dams were treated preterm or term.

Culture of equine trophoblastic vesicles in vitro.

Trophoblastic vesicles have been used to study early embryonic development and maternal recognition of pregnancy in domestic animals. The purpose of this study was to characterize the formation of trophoblastic vesicles from Day-12 to Day-16 equine conceptuses. Conceptuses (n = 19) were collected nonsurgically from mares, the capsule was removed, and the conceptus (trophoblast and inner cell mass) was dissected into 2- to 4-mm fragments. Conceptus fragments were cultured in either Ham's F10 (HF10) or Minimum Essential Media (MEM) with 10% fetal bovine serum (FBS) in 24-well plates. Plates were incubated at 37 degrees C in a humidified atmosphere of 90% N(2), 5% O(2), and 5% CO(2) and were examined at 48 and 96 h for the number and diameter of trophoblastic vesicles formed. There was no significant difference (P > 0.1) between HF10 and MEM in the diameter of trophoblastic vesicles at 48 or 96 h of culture. There was, however, a significant increase (P < 0.01) in the number of trophoblastic vesicles per conceptus between 48 and 96 h of culture for HF10 and MEM. The mean diameters of trophoblastic vesicles after 96 h in culture were 396.4 +/- 19.9 mum and 415.0 +/- 12.0 mum for HF10 and MEM, respectively. Histologic sections of trophoblastic vesicles revealed a bilaminar structure consistent with the presence of trophectoderm and endoderm. Squash preparations of trophoblastic vesicles contained cells similar to those recognized in squash preparations of fresh conceptuses. Areas of increased cell density that resembled the inner-cell mass were seen in both fresh trophoblastic vesicles and in sections of trophoblastic vesicles. Equine trophoblastic vesicles may be useful to further the study of early embryonic development and pregnancy recognition in mares.

Survival of equine embryos transferred to normal and subfertile mares.

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mare's uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.

Pregnancy rates at Days 2 and 14 and estimated embryonic loss rates prior to day 14 in normal and subfertile mares.

Pregnancy rates at Days 2 and 14 postovulation were determined for 15 normal mares and 15 subfertile mares. Embryonic loss rates were estimated by the difference in the Day 2 and Day 14 pregnancy rates. Mares were artificially inseminated with the pooled ejaculates from three stallions, and the embryonic vesicle was detected with ultrasonography at Days 9, 10, 12 and 14. Mares were short-cycled with prostaglandin F(2) alpha (PGF(2alpha)) and rebred to the same stallions, and the Day 2 pregnancy rates were determined by recovery of cleaved ova (embryos) from the surgically excised oviducts. Significantly more (P < 0.01) normal versus subfertile mares were pregnant at Day 14 (12 15 vs 3 15 ). There was no significant difference in the Day 2 pregnancy rate for normal versus subfertile mares (10 14 vs 11 14 ). There were no significant differences (P > 0.5) in the mean number of blastomeres per embryo or in the mean diameter of embryos recovered at Day 2 from normal or subfertile mares. The estimated embryonic loss rate was significantly lower (P < 0.01) for normal verusus subfertile mares (0 10 vs 8 11 ). Fertilization rates were similar for normal and subfertile mares; however, subfertile mares had a higher embryonic loss rate prior to Day 14 postovulation.

Avulsion of the bovine prepuce from its attachment to the penile integument during semen collection with an artificial vagina.

Avulsion of the prepuce (AP) from the penile integument during intromission into an artificial vagina (AV) is the most common injury involving the penis and prepuce of bulls collected for the production of frozen semen at artificial insemination (AI) centers. We observed clinical cases at two AI centers. Because temporary loss of seminal collections from genetically superior bulls occurs and the possibility of impotency exists, the economical loss from AP is significant. Surgery, rather than a nonsurgical procedure, is the preferred method for repair of AP if the diagnosis occurs on the same day as the injury. After injury, seminal collection is avoided. The bull can then begin a conditioning program prior to returning to a normal semen collection schedule. A specific etiology for AP remains undetermined; however, many variables potentially involved in its occurrence are known. Bulls with good libido following 4 to 5 yr of sexual inactivity are particularly susceptible to AP. No genetic predisposition was observed. At present, we believe that AP is caused by many factors and that implementing preventive measures does help.

Induction of abortion in mares with equimate: effect on secretion of progesterone, PMSG and reproductive performance.

Thirty-two light-horse mares were confirmed to be pregnant and assigned to one of four treatments: (1) injected with 250 micrograms of Equimate on day 70 and again on day 77 if abortion had not occurred; (2) injected with 250 micrograms of Equimate on day 70 and every 24 hr until abortion occurred (maximum four injections); (3) injected with 250 micrograms of Equimate on day 70 and every 12 hr until abortion (maximum eight injections); and (4) injected with 250 micrograms of Equimate once only on day 35 of gestation. Mares were observed four times daily for incidence of abortion or side effects. Estrual behavior was monitored daily and follicular activity either daily or every third or fourth day until estrus and ovulation. Samples of jugular blood were obtained at 0600 hr day -1 and every 6 hr until the first day of estrus after abortion, or for 2 weeks after abortion if estrus had not occurred, or for 1 week after treatment for mares that had not aborted. A single injection of Equimate terminated pregnancy in all but one mare injected on day 35, but none of the mares given an injection on day 70 and 77 aborted. Multiple injections of Equimate beginning on day 70 of gestation terminated pregnancy in all mares. Fewer (P less than .05) injections were required for abortion of mares injected daily. During the immediate week post-treatment, concentrations of progesterone decreased (P less than .05) in all mares injected with Equimate, but overall concentrations were greater (P less than .05) for mares injected once on day 70 than for those in the other three groups. Equimate did not affect secretion of PMSG in the day 70 group of mares. Estrus and ovulation after abortion were delayed (P less than 0.05) for mares injected daily or twice daily beginning on day 70 compared to those injected once on day 35. Thus, rebreeding of these mares during the same breeding season in which they were aborted would be difficult.

Rates of diagnosis of six diseases of Holstein cows during 15-day and 21-day intervals.

The lactations of 3,172 Holstein dairy cows were divided into intervals of 15 and 21 days. The numbers of new diagnosis of metritis, cystic ovary, miscellaneous reproductive disorders, mastitis, left displaced abomasum, and milk fever in each interval were counted. The numbers of cows at risk in each interval were also counted. A cow was at risk if she was still milking, in the herd, and a diagnosis had not already been made for that condition in that lactation. A cow was also only at risk of a reproductive diagnosis if she was not pregnant. For each interval, the incidence rate of new diagnosis was calculated by dividing the number of new diagnoses by the number of cows at risk.

Temperature of the artificial vagina and its effect on seminal quality and behavioral characteristics of stallions.

Stallion semen was collected, using artificial vaginas at 44 to 46, 48 to 50, and 52 to 54 C, to study the effects of temperature on seminal quality and sexual behavior. The temperature of the artificial vagina had no significant effect on motility, gel volume, gel-free seminal volume, total seminal volume, pH, number of mounts per ejaculate, total time to ejaculation, or seminal temperature. Spermatozoa were collected, then exposed to water-bath temperatures of 38, 45, 49, or 53 C for 1 minute. Mean motility was similar after exposure to temperatures of 38 or 45 C, but exposure to temperatures of 49 or 53 C resulted in a significant decrease (P < 0.05) in spermatozoal motility with each increase in temperature. Thus, it was concluded that spermatozoa exposed to temperatures greater than 45 C may be irreversibly damaged.

Metabolic abnormalities associated with rupture of the urinary bladder in neonatal foals.

Rupture of the urinary bladder was diagnosed in 4 neonatal male foals. Marked hyponatremia, hypochloremia, and hyperkalemia developed in all 4 cases, and the electrolyte abnormalities were considered to be of potential diagnostic value. Blood urea nitrogen concentration was normal or only slightly higher than normal in 3 of 4 cases and therefore was not a reliable diagnostic test.

Association of parturient hypocalcemia with eight periparturient disorders in Holstein cows.

The association between parturient hypocalcemia and 8 periparturient disorders (dystocia, retained fetal placenta, metritis, left displaced abomasum, foot problems, ketosis, mastitis, and coliform mastitis) was evaluated in 33 Holstein dairy herds (2,190 cows) that calved from March 1981 through February 1982. There were highly significant (P less than 0.0001) associations between parturient hypocalcemia and dystocia, retained fetal placenta, ketosis, and mastitis. The odds ratios (multiplicative increases in occurrence) for these associations were 6.5, 3.2, 8.9, and 8.1, respectively. There was a significantly (P less than 0.03) association between parturient paresis and coliform mastitis (odds ratio, 9.0). There was a tendency for association with left displaced abomasum (odds ratio, 3.4; P less than 0.06).

A randomized clinical trial of the effect of the time of administration of cloprostenol on detention of estrus in dairy cattle.

We performed a clinical trial to study whether cows with palpable corpora lutea treated with cloprostenol by the farmer on the morning following examination (median time 0630 hours) were seen in heat and conceived more frequently than cows treated by the veterinarian immediately following the reproductive examination (median time 1210 hours).There were no significant differences between the two groups in the proportions of cows seen in estrus or fertility at the induced estrus.The proportions of animals that had the time of onset of estrus recorded and were seen in estrus during working hours (0800-1600 hours) were also not significantly different between the veterinary-treated and farmer-treated groups.Under the conditions of this study, there was no reason to prefer prescribing early morning administration of cloprostenol by farmers over immediate administration of the drug by the clinician.

Induction of parturition in mares.

Thirty-seven induced parturitions involving thirty-one mares were studied over a 2-year period using various doses of oestrogen and oxytocin. The time of appearance and degree of expression of the major clinical signs of parturition, and the time for completion of delivery and the passage of the placenta were significantly influenced by increasing doses of oxytocin. Oestrogen was useful in softening and relaxing the cervix when it was tight, but was not essential to induction when the cervix was already soft and dilating. Fertility was not adversely affected by induced parturition.

Hormonal changes in the mare and foal associated with oxytocin induction of parturition.

Oxytocin was used to induce parturition in 6 mares and to determine the hormonal changes occurring during and for 72 h after parturition in the mares and their foals. Normal, healthy foals were born shortly (about 34 min) after a single i.m. injection of 40 or 60 i.u. oxytocin. There was no retention of fetal membranes and all mares produced ample milk. Immediately after foaling oestrogen and progesterone levels in the dam were 36 and 29% of preinjection means while the total corticoid levels remained relatively constant throughout the sampling period. The systemic levels of total oestrogens, progesterone and total corticoids immediately after birth were significantly (P less than 0.01) greater for the foal than the dam, and all declined in the foal throughout the 72-h sampling period. The levels of oestrogens and progesterone were greater (P less than 0.05) in the umbilical vein than umbilical artery, indicating the endocrine function of the placenta. However, total corticoids were greater in the umbilical artery than in the umbilical vein. The corticoid level in the jugular vein of the foal at birth was greater than that of the umbilical vein suggesting a fetal contribution to the total corticoid level.

Oestrogen excretion in mares in relation to various reproductive states.

The rate of urinary oestrogen excretion was studied in four ovariectomized mares. In two animals ovariectomized when urinary oestrogen concentration was high, levels fell to a minimun within 48 hr. Intramuscular injection of oestradiol-17beta produced maximum urinary oestrogen levels within 3 to 6 hr and a return to minimum detectable levels within 24 hr. The major urinary oestrogen in mares was oestrone. In nine maiden mares studied between September and June, the cycle, the durations of dioestrus and oestrus, and the time of ovulation were similar in autumn (September to December) and spring (January to June). All nine mares had periods of anoestrus and 'shallow' and 'deep' forms of this condition were observed. In all cyclic mares, urinary oestrogen excretion showed a gradual rise from dioestrous levels beginning 6 to 7 days before ovulation. A rapid and highly significant (P less than 0-001) fall in oestrogen excretion occurred on the day of ovulation and the day after. Maximum urinary oestrogen levels were twice as high in spring than in autumn cycles.

Recovery and evaluation of embryos from normal and infertile mares.

To evaluate embryo transfer as a possible method to circumvent infertility in mares, embryos from 14 normal and 14 infertile mares were collected three times and examined. Fewer flushes (p less than 0.05) from normal than infertile mares (1/42 vs 9/42) contained only abnormal embryos whereas more flushes (p less than 0.05) from normal than infertile mares contained one or more normal embryos (28/42 vs 8/42). More flushes (p less than 0.05) from normal than infertile mares contained embryos (29/42 vs 17/42). The embryo diameters (mm) at either day-7 or day-8 post ovulation were greater (p less than 0.01) for normal than infertile mares (day 7: 07 +/- 0.08 vs 0.3 +/- 0.07; day 8: 1.1 +/- 0.18 vs 0.7 +/- 0.23). Six of the 10 (60%) flushes that contained only abnormal embryos were recovered from ares with positive uterine cultures or moderate to severe endometritis. The embryos recovered from normal mares were greater in quantity and better in quality.

Atypical actinobacillosis granulomas in cattle.

Actinobacillus lignieresii is well recognized as the cause of "wooden tongue." We have encountered Actinobacillosis as the cause of granulomas in unusual locations. These lesions have been confused with neoplasms, nasal polyps, or exuberant granulation tissue. Four such cases constitute this report.

Semen characteristics in thyroidectomized stallions.

Three Quarter-horse stallions were surgically thyroidectomized at 18 months of age. Weekly semen collections were made from these and three control stallions from 25 to 39 months of age. Although the induced state of hypothyroidism in young stallions has a depressing effect on libido and general animation and can be corrected by thyroprotein supplementation, semen characteristics, testicular histology and fertility are not affected by the operation.

Chlorpyrifos (Dursban 44r) toxicity in dairy bulls.

The toxicity of Dursban 44 (chlorpyrifos) in dairy bulls in two large Northeastern A.I. bull centers is reported. One hundred eighty-five (185) bulls were treated at the first center and two hundred thirty-two (232) at the second. The application of this product resulted in the death of seven and one bulls, respectively. The knowledge and experience gained at the first center resulted in rapid diagnosis and specific treatment at the second and was felt to be a significant reason for the lower death rate. Typical, clinical signs of organophosphate poisoning were not exhibited in these two outbreaks. Specific diagnosis, treatment and the course of chlorpyrifos poisoning in Holstein bulls are discussed.