The laparoscope in follicular oocyte collection and gamete intrafallopian transfer and fertilization (GIFT).

Laparoscopic recovery of bovine follicular oocytes was studied. The collection of oocytes from the superovulated bovine ovary was maximized by standardizing the collection technique. The technique was highly successful, with a 79% oocyte recovery rate of the follicles aspirated. Collected oocytes were transferred to the inseminated recipient's oviduct with a minimum of trauma through the laparoscope. This gamete intrafallopian transfer and fertilization (GIFT) resulted in multiple embryo recovery in the cow. Oviductal catheterization and the potential of GIFT are described and discussed.

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility.

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.

Progesterone in bovine milk fat.

The progesterone (P(4)) concentration (ng/5 mul) in the fat portion of cow's milk was measured on days 0, 12, 20, 24, and 30 after insemination in an effort to assess the ability of this analysis to judge the reproductive status of dairy cows. Pregnancy was determined by rectal palpation at approximately 40 days after insemination. The mean P(4) concentration (ng/5 mul) +/- SD in the pregnant cows (n=17) was 0.14+/-.07, 1.31+/-.36, 1.41+/-.52, 1.22+/-.21, and 1.30+/-.43 on post-insemination days 0, 12, 20, 24, and 30 respectively. At these same intervals the P(4) concentration in the non-pregnant (NP) cows (n=18) was 0.14+/-.06, 1.26+/-.37, 0.56+/-.52, 0.57+/-.42, and 1.08+/-.59. On days 20 and 24 after insemination, mean P(4) levels were significantly (P<.001) elevated in the pregnant cows. It was concluded that an accurate assessment of the reproductive status of a cow, milk samples from days 0, 20, and 24 post-insemination would have to be analyzed for milkfat P(4) concentrations. In order to determine the percentage of cows inseminated out of the periestrual period, milkfat P(4) concentrations were ascertained in milk collected from 165 cows at the time of insemination. Cows that conceived (n=38) had a mean milkfat P(4) concentration at the time of insemination of 0.16+/-.09. The upper limit for P(4) in the milkfat at the time of insemination in cows that conceived was calculated to be 0.43 ng/5 mul milkfat. Subsequently, it was found that of the 14 cows that had P(4) levels above this upper limit at time of insemination, nine were inseminated at a period other than in the periestrual period and five were inseminated while already pregnant.

Spermagglutinins in serum and seminal fluid of bulls and their relationship to fertility classification.

Twenty-five satisfactory and 25 unsatisfactory potential breeder bulls were examined with a modified Shulman spermagglutination test for the presence of spermagglutinating antibodies in their serum and seminal fluid. This was done to determine whether antibodies to spermatozoa were a contributing factor to classification of the bull as an unsatisfactory potential breeder. No correlation was found between classification as an unsatisfactory potential breeder and the presence of spermagglutinating antibodies. In addition, indirect fluorescent antibody tests were performed to detect antisperm antibodies in the serum and seminal fluid of the bulls. Again, no correlation was found between antisperm antibodies and breeding soundness classification. Finally, there was no correlation between the age of the bulls and occurrences of spermagglutination antibodies in serum or seminal fluid.

Thyroidal and prolactin secretion in agalactic mares.

A study was designed to examine serum concentrations of prolactin (PRL) and iodothyronines before and after thyrotropin releasing hormone (TRH) administration to agalactic (n = 26) and normally (n = 8) lactating mares. Two mg TRH was given intramuscularly (i.m.) twice daily on Day 1 (day of delivery) through Day 5. Jugular venous blood was collected on Days 1 and 5 before TRH (time 0) and at 1 and 3 h post-TRH. Basal serum concentrations of thyroxin (T(4)) were different (P < 0.05) on Day 1 (1.87 vs 1.37 mug/dl) and Day 5 (1.72 vs 1.13 mug/dl) in the normal mares and agalactic mares, respectively. There was no difference in the T(4) response to TRH. While basal serum concentrations of triiodothyronine (T(3)) were not different, agalactic mares responded with greater (P < 0.05) serum concentrations T(3) to TRH on Day 1. Following linear regression of the PRL response to TRH, slope of the lines between groups did not differ; however, elevations were significantly (P < 0.05) greater (1.79 vs 1.28 ng/ml) in control mares compared with agalactic mares, respectively, on Day 1.at 1 h post-TRH. A similar difference existed at time 0 and 1 h on Day 5. Consequently, agalactic mares had reduced basal serum T(4) values; the PRL data leads us to suggest that secretion of this hormone may be insufficient in agalactic mares.

Comparison of Tifton 85 and Coastal bermudagrasses for yield, nutrient traits, intake, and digestion by growing beef steers.

A study was undertaken to compare Tifton 85 (T85) and Coastal (CBG) bermudagrasses for effects of cultivar and age at harvest on yields of DM and digestible DM, in vitro digestion, nutrient content, cell wall composition, in situ digestion kinetics, and feed intake and digestion by growing beef steers. In Exp. 1, T85 and CBG forages staged for growth in May or July of 1993 were harvested at 3, 4, 5, 6, 7, and 8 wk from subplots. Tifton 85 bermudagrass had 7.1% greater DM yield, 18.2% higher (P < .05) digestible DM yield, and 7.1% greater IVDMD than CBG, and, after 5 wk of forage growth, IVDMD of both T85 and CBG decreased with increased age at harvest (P < .05). In Exp. 2, T85 and CBG forages staged for growth in July 1997 were harvested at 2, 3, 4, 5, 6, and 7 wk from subplots. Even though T85 had higher concentrations of NDF and ADF than CBG, T85 had 34.1% higher DM yield, 47.9% higher digestible DM, 55.0% higher digestible NDF, 91.7% higher digestible ADF, greater IVDMD, in vitro NDF and ADF disappearances, and higher in situ DM and NDF digestion (P < .05). Coastal bermudagrass had higher concentrations of lignin and lower concentrations of total neutral sugars, arabinose, glucose, and xylose than T85 (P < .05). In vitro digestibilities of DM, NDF, and ADF were lower and concentrations of ADF and lignin were greater for 7- vs 6-wk harvests of both T85 and CBG (P < .05). In Exp. 3, T85 and CBG forages staged for growth in July 1997 were harvested as hay at 3, 5, and 7 wk from .8-ha pastures and fed to 36 individually penned growing beef steers (initial BW = 244 kg) to quantify ad libitum intake without supplementation. Tifton 85 bermudagrass had lower concentrations of lignin and ether-linked ferulic acid and greater concentrations of NDF, ADF, hemicellulose, and cellulose than CBG (P < .05). Steers fed T85 had higher (P < .05) digestion of DM, OM, NDF, ADF, hemicellulose, and cellulose than steers fed CBG. Digestion of NDF, ADF, hemicellulose, and cellulose decreased (P < .05) with increased age at harvest for both cultivars. In conclusion, T85 produced more DM and had more digestible nutrients in vitro, in situ, and in vivo than CBG, and 3 and 5 wk of growth would be recommended ages to harvest either cultivar.

Luteinizing hormone secretion before and after ovariectomy in prepubertal and pubertal beef heifers.

The effects of sham ovariectomy (SOVX) or ovariectomy (OVX) on (1) basal luteinizing hormone (LH) concentrations, (2) number of LH peaks per hour and (3) magnitude of LH peaks were determined in prepubertal and pubertal beef heifers. Basal LH concentrations were not influenced by SOVX of prepubertal or pubertal heifers. However, after OVX, basal LH concentrations increased between 168 and 192 hr in prepubertal heifers and between 72 and 80 hr in pubertal heifers. The number of LH peaks per hour was not influenced by SOVX but OVX caused an increase in the number in both prepubertal and pubertal heifers, and the increase occurred sooner after OVX in pubertal than in prepubertal heifers. The magnitude of LH peaks was similar for both prepubertal and pubertal heifers prior to SOVX and OVX. Magnitude of LH peaks was unchanged after SOVX in prepubertal and pubertal heifers, but increased within the first 24 hr after OVX in pubertal heifers and within 168 to 192 hr after OVX in prepubertal heifers. These data indicate that inhibition of LH secretion by ovaries occurred in both prepubertal and pubertal beef heifers. The time-related differences between prepubertal and pubertal heifers in basal LH concentrations, number of LH peaks per hour and magnitude of the LH peaks after OVX may reflect a difference in endocrine environment in these two reproductive states. However, this hypothesis was not tested in this experiment.

Influence of endophyte-infected tall fescue on serum prolactin and progesterone in gravid mares.

Thirty mares in late gestation were used in a 3-yr study to assess effects of the tall fescue endophyte Acremonium coenophialum on serum prolactin (PRL) and progesterone. Two paddocks of each treatment, 0 or 100% infected 'Kentucky 31' tall fescue, were grazed by the mares for 21 d. Blood was collected three times per week until parturition. At 7-d intervals, mares were challenged with thyrotropin-releasing hormone (TRH) while grazing and blood was collected postinjection. Mares grazing 100% infected tall fescue (E+) had decreased serum PRL compared with mares grazing the 0% infected tall fescue (E-) in 2 of 3 yr. Within 8 d postgrazing, serum PRL for E+ mares equaled or surpassed values of the E- mares. Serum PRL was not different during the 3rd yr. In response to TRH, serum PRL rate of increase was similar between treatments but remained elevated (P less than .01) in the E+ mares at the 180-, 240-, and 300-min sample times. Serum progesterone was lowered (P less than .05) by E+ but increased to control values within 10 d postgrazing. It is concluded that serum PRL and progesterone in the gravid mare were decreased by the presence of A. coenophialum in 'Kentucky 31' tall fescue grass but normal levels were reestablished within 2 to 3 wk.

Effect of induced prostatic infection on semen quality in the dog.

Chronic Escherichia coli prostatitis was induced in 6 dogs, with 5 sham-operated dogs serving as controls. Semen volume and sperm motility, concentration, and appearance were measured 3 times (every other day) before surgery and 3 times during the 3rd and 5th week after surgery. When values for these collections were compared, using a 2-factor analysis of variance, no significant differences (P greater than 0.05) were found. However, the percentage of normal sperm decreased markedly (from 92% to 61% to 64%) in 2 infected dogs because of an increase in primary (from 6% to 32%) or secondary (from 2% to 28%) sperm abnormalities. Testes from both of these dogs were histologically normal.

Morphometric studies of the bovine uterus: microscopic lesions and retrospective reproductive history.

A survey of reproductive tracts from nonpregnant cows at an abattoir was conducted. Reproductive tracts from 98 cows were studied microscopically and evaluated with a grading system to determine the severity of pathologic changes. Inflammatory reaction (type and distribution), fibrosis, gland morphology, and appearance were measured, quantified, and scored (1 to 4). Category 1 endometrium (normal) was found in 18 cows, category 2 in 23, category 3 in 34, and category 4 in 23. The category of endometrium was then compared with the retrospective reproductive status, and it was found that reproductive problems had occurred in 6 cows (33.3%) in category 1, in 13 cows (56.5%) in category 2, in 25 cows (73.5%) in category 3, and in 21 cows (91.3%) in category 4. Cervicitis was found in 43 (43.8%) genital tracts; 16 cows (16.3%) had salpingitis. Ovarian lesions were not observed. Serum samples were tested for certain antibodies. Only 1 was seropositive for brucellosis, but 61 of 81 sera had leptospira titers greater than 1:100 and 1:50 (hardjo). The serovar hardjo was the most prevalent. All sera had neutralizing titers against parainfluenza type 3 virus; 74.2% and 56.7% of the sera had neutralizing antibodies against bovine viral diarrhea virus and infectious bovine rhinotracheitis virus, respectively.

Association of blood cholesterol with occurrence of fat necrosis in cows and tall fescue summer toxicosis in steers.

Factors associated with fat necrosis in cows and tall-fescue summer toxicosis in steers were studied. In the cow study, fescue pastures were fertilized, using 3 rates of N: high N (703 to 483 kg and 0 kg of N/ha/year from broiler litter in 1972 to 1974 and 1975, respectively), moderate N, and low N (224 and 74 kg of N/ha/yr from NH4NO3, 1972 to 1975, respectively). Bermuda grass pastures were fertilized at 2 rates of N: moderate N and low N (280 and 20 kg of N/ha/year from NH4NO3, 1972 to 1975, respectively). Fat necrosis developed only in cows grazing tall fescue, with an occurrence of 60%, 8%, and 3% for high-N, moderate-N, and low-N pastures, respectively. Cows grazing the high-N fescue, and to some extent those grazing the moderate-N fescue, had clinical signs of summer fescue toxicosis. Plasma cholesterol concentrations were lowest in cattle grazing the high-N fescue, averaging 114 mg/dl, followed by 134 and 127 mg/dl in cattle grazing the moderate-N and low-N fescue, respectively. In the steer grazing study, 24 paddocks of 0.49 ha each were seeded with tall-fescue lines G1-307 or G1-306 or with tall-fescue cultivars, KY-31 or Kenhy. All paddocks were fertilized with 170 kg of N/ha/year. Serum cholesterol concentrations were lower in steers grazing on G1-307 than in steers grazing on G1-306 or cultivars. Serum total lipids followed a similar trend, with a positive correlation (r = 0.49) between cholesterol and total lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a once-daily suckling program on gastrointestinal parasitism in Angus cows and calves.

Over a 2-year period involving a total of 168 Angus cows and their calves, 2 studies were conducted to evaluate the effect of once-daily suckling on cow reproduction, calf growth, and gastrointestinal parasitism. Experiment I compared 2 restricted suckling (RS) groups with 1 normal suckling (NS) group. Each group consisted of 24 cows with their calves (24 cows-calves/group = 24 cows and 24 calves/group). In the RS groups, suckling by the calves was restricted to once a day for 45 days, and then the calves were allowed to suckle normally throughout the remainder of the experiment. In the NS group, the calves suckled normally throughout the experiment. Experiment II compared 2 groups of RS cow-calves (24 cow-calves/group) with 2 groups of 24 NS cow-calves/group. In experiment I, there were no differences (P less than 0.05) in pregnancy rate or in the average number of nematode eggs/g of feces (epg) between the cows from the 2 treatment groups. The RS calves consistently had higher epg counts than did the NS calves during the 45 days of RS (169 vs 62 epg, P less than 0.05) and from the time of their return to pasture until weaning (266 vs 186 EPG, P = 0.27). The RS calves also had a higher average coccidia oocyst count during the RS period only. In experiment II, RS cows had a higher pregnancy rate (87.6% vs 66.0%, P less than 0.05) and a shorter postpartum interval (83 vs 101 days, P less than 0.05) than did NS cows.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicular cystic ovaries and cystic endometrial hyperplasia in a bitch.

Treatment of cystic ovaries in a bitch was unsuccessful with human chorionic gonadotropin, but aspiration of the follicles at laparotomy was successful. Follicular aspiration is described, with special emphasis on preventing bursal adhesions. Aspiration of persistent follicles is a simple treatment for cystic ovarian disease that is nonresponsive to parenterally administered hormones. The persistent estrus associated with cystic follicles is a primary cause of cystic endometrial hyperplasia (CEH) and severe cases of CEH are associated with infertility. Rapid intervention (surgical or hormonal) will limit the severity of CEH and, therefore, decrease infertility in breeding bitches.

Infertility in a ewe as a result of ovotestis.

A ewe with apparently normal estrous cycles was unable to conceive as a result of a single ovotestis. The ovotestis was diagnosed on the basis of results from laparoscopic examination, human chorionic gonadotropin stimulation, and histologic examination of the gonad. The report reinforces the necessity of considering all causes of infertility in affected sheep, and not simply infective causes.

Prostaglandin F2 alpha metabolite levels in normal and uterine-infected postpartum cows.

The stable metabolite of prostaglandin F2 alpha,15 keto-13,14-dihydroprostaglandin F2 alpha (PGFM), was measured from peripheral blood samples collected at specified intervals postpartum from 7 normal dairy cows and 4 cows with apparent endometritis. Plasma PFGM levels were significantly (P less than .05) elevated for the first 5 days postpartum in the cows with endometritis (ranging from 4.0 to 5.0 ng/ml) compared to the controls (approximately 1.0 ng/ml). Beyond 5 days postpartum, plasma PGFM levels were not significantly different and decreased to approximately 0.4 ng/ml by day 13 in both groups. Time to uterine involution was not different between groups (less than 30 days). Therefore, uterine infections in cows during the puerperium was associated with elevated circulating PGFM levels. These findings and the observation that PGF2 alpha is not uterotonic in the puerperal cow do not suggest a therapeutic use of PGF2 alpha in order to evacuate the uterus.

Restraint.

The object of restraint is to limit, restrict, and keep under control the animal's movement while performing different procedures. The author discusses the importance of evaluating the horse and its owner prior to employing any type of restraint, various concepts of restraint, and examples of situations in which restraint may be required.

Fine structure of equine oocytes matured in vitro for 15 hours.

Transmission electron microscopy (TEM) was used to evaluate the fine structure of equine oocytes cultured in vitro. Oocytes obtained by follicular aspiration were cultured for either zero or 15 hr. After treatment oocytes were processed either by light microscopy (nuclear evaluation) or TEM (cytoplasmic evaluation). Those oocytes cultured for 15 hr were incubated in modified TCM 199 with 15% (v/v) mare serum (day of ovulation) at 39 +/- 0.2 degree C. Evaluation using TEM revealed that cortical granules were present in all oocytes. However, zero-time oocytes contained few cortical granules, and these were scattered throughout the cytoplasm, whereas 15 hr oocytes contained numerous cortical granules primarily found in very close proximity to the oolemma. Further ultrastructural analysis of both groups revealed organelle structure similar to that previously described for in vivo matured equine oocytes. Evaluation of nuclear maturity (lacmoid stain) showed that 15 hr of culture resulted in significant numbers of oocytes at metaphase II (8/17; 47%). These data demonstrate that oocytes cultured for 15 hr in modified TCM 199 with 15% mare serum (day of ovulation) are mature with respect to nuclear configuration and cortical granule migration and, therefore, would be appropriate candidates for in vitro fertilization.

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.

Galactosyltransferase activity is restricted to the plasma membranes of equine and bovine sperm.

beta 1, 4-Galactosyltransferase (GalTase) is localized to the plasma membrane of mouse sperm, in which it mediates the binding of sperm to glycoconjugate residues in the egg zona pellucida. In this study, the presence of subcellular distribution of sperm GalTase were determined in two other mammalian species that yield sufficient sperm for subcellular fractionation. Equine and bovine semen were collected, and the plasma membranes (PM), outer acrosomal membranes (OAM), and inner acrosomal membranes (IAM) were sequentially removed. The purities of the isolated membrane preparations were determined by transmission electron microscopy and found to be greater than or equal to 90%, 96%, and 98% for equine PM, OAM, and IAM, respectively, and greater than or equal to 80%, 94%, and 97% for bovine PM, OAM, and IAM, respectively. GalTase activity was assayed under optimal conditions in all membrane preparations and was preferentially localized to the isolated PM both in equine and in bovine spermatozoa. The selective localization of GalTase to the sperm PM in two other species suggest that it may serve as a generalized gamete receptor during initial sperm-egg binding in mammals.