L.F. Archbald

Comparison of a solid-phase, no-extraction radioimmunoassay for progesterone with an extraction assay for monitoring luteal function in the mare, bitch, and cow

Abstract Equine, canine, and bovine plasma samples were assayed for progesterone (P 4 ) using extracted plasma in a liquid-phase assay and unextracted plasma in a solid-phase 125 I direct assay developed for human serum. The direct assay was also used to monitor P 4 levels in defatted milk. Results indicated that the direct-assay method was as reliable as the extraction assay for monitoring changes in plasma P 4 during the estrous cycle and early pregnancy. The regression equation specifying the relationship for the two methods was exponential. In this study, correlation coefficients from data subsets ranged from 0.93 to 0.99. The direct assay for P 4 did result in higher values than the extraction assay in plasma from diestrous animals. The use of diluted standards with stripped plasma from the species being assayed gave values that correspond more closely to the extraction assay. The comparisons in this study indicate that the direct radioimmunoassay (RIA) method offered a convenient, reliable method for monitoring luteal function in the species that were evaluated.

Comparison of once daily and twice daily FSH injections for superovulating beef cattle.

Twelve cycling Angus-based crossbred cows were used in a crossover experimental design to evaluate two different injection schedules using Follicle Stimulating Hormone (FSH) for superovulating donor cattle. Females randomly assigned to Treatment (A) were given twice daily FSH injections of 5 mg each (12 hours apart) for five consecutive days starting on day 10 of the estrous cycle while those in Treatment (B) received the same daily dose level of FSH, except it was given in a 3.2% protein gelatin carrier vehicle and administered on a once daily injection schedule. Animals in both Treatments (A) and (B) were each given a 30 mg dose of commercially available prostaglandin-F(2alpha) agent 48 hours after the first FSH injection. Cows in estrus were initially handmated to a fertile bull then artificially inseminated 12 hours later with two units of frozen semen. All 12 animals (100%) given twice daily FSH injections and 11 of the females (91.6%) administered once daily FSH injections exhibited standing estrus within 5 days following injection of the luteolytic agent. On day 7 or 8 after the onset of standing estrus a laparotomy was performed to observe ovarian structures. When the superovulation response was evaluated, the mean number of corpora lutea per ovary ranged from 2.9 in the twice daily injection group to 4.1 in the once daily injected group. Unexpectedly, the once daily treated group had significantly more corpora lutea per animal (8.1 vs. 6.4) than those in the twice daily treated group. In addition, mean ovarian size score per animal increased significantly when pre-treatment scores were compared to those recorded following FSH treatment (laparotomy) in both Treatment (A) and (B), however, the post-treatment ovarian size scores were not different between these groups. When evaluating post-treatment follicular development, the once daily injection group had significantly more smaller follicles (<10 mm) and a greater number of ovulatory size follicles (>10 mm) than the twice daily injection group. Furthermore, viable appearing embryos were recovered from both treatment groups and no adverse reactions were observed with the gelatin carrier vehicle in Treatment (B). Since the once daily FSH injection schedule resulted in a superovulatory response equal to or greater than the twice daily FSH injection schedule, this approach to superovulation should not be overlooked by those involved in bovine embryo transplantation.

Effect of the bovine viral diarrhea (BVD) virus on preimplantation bovine embryos: A preliminary study

Five crossbred-Holstein cows, approximately three to seven years of age, were superovulated using pregnant mares serum gonadotropin (PMSG) and prostaglandin F2α (Prostin F2α). At the induced estrus, each cow was artificially inseminated with frozen semen. Seven days after insemination, the lumen of the right uterine horn of each cow was inoculated with BVD virus in Eagles minimum essential tissue culture medium, and the lumen of the left horn was infused with tissue culture medium only. Three days later, each cow was subjected to midventral laparotomy under general anesthesia and embryos were collected. A total of 22 embryos were recovered; 12 were from infected uterine horns and ten were from non-infected uterine horns. All embryos from the non-infected uterine horns were in the late blastocyst stage without the zona pellucida. Of the embryos collected from the infected uterine horns, eight of 12 (66.6%) still possessed zona pellucida and appeared in a degenerative state. The remaining four embryos were morphologically similar to those from the non-infected uterine horns. Electron microscopic examination of the degenerated embryos from the infected uterine horns demonstrated the presence of a structure which morphologically resembled the BVD virus. The results of this preliminary study indicate that the BVD virus within the uterine horns may interfere with normal development of preimplantation bovine embryos. Therefore, it is proposed that the BVD virus could adversely affect early stages of gestation in the cow, resulting in infertility.

Effect of gonadotropin-releasing hormone and prostaglandin F2α on reproduction in postpartum dairy cows

Abstract Thirty dairy cows serving as the treated group (Group A) were injected intramuscularly with 100 mcg gonadotropin-releasing hormone (GnRH) at 10 to 16 days postpartum followed by 25 mg prostaglandin F 2 α (PGF 2 α) 14 days later. Twenty-nine herdmate dairy cows (Group B) serving as controls were treated in a similar manner using saline injections rather than GnRH or PGF 2 α treatments. Only cows without obvious uterine infection were assigned to the experimental groups, and any uterine pathology that developed during the treatment interval was treated accordingly following the experimental period. Internal genitalia were evaluated via rectal palpation prior to each injection. Blood samples were collected for progesterone analysis before each injection and at 30 hours following the PGF 2 α or the second saline injection. Experimental animals were artificially inseminated at the first detected postpartum estrus starting 35 to 40 days following calving. Results indicated evidence of enhanced cyclicity when Group A cows were compared with those in Group B. However, there were no significant differences between the two groups for interval to first observed estrus, interval to first serive, first serive pregnancy rate, services per pregnancy and days open. Furthermore, no difference in the incidence of follicular or luteal cysts, incidence of repeat breeders or number of reproductive culls was observed. From observations in this study, the GnRH and PGF 2 α treatment scheudule might not be economically beneficial in lactating dairy cows as long as reproductive tract abnormalities are promptly diagnosed and subsequently treated by the attending practitioner.

Luteal function of induced corpora lutea in the bitch

Nineteen anestrous bitches with a mean of 22 kg body weight and ranging from 2 to 4 years of age were induced to exhibit estrus and ovulate using PMSG and HCG. Twelve days after the first day of estrus, bitches were assigned to four treatment groups. Group (A) consisted of six bitches, Group (B) of five bitches and Groups (C) and (D) of four bitches each. At this time, bitches in Groups (A), (B) and (C) were laparotomized and those assigned to Groups (A) and (B) were bilaterally hysterectomized leaving the cervix and oviducts intact. Although bitches in Group (C) were laparotomized, they were not hysterectomized. Group (D) bitches were not subjected to any surgical procedures. Homologous uterine extract was prepared from each bitch in Group (A) and administered intramuscularly beginning on day 25 (day 0=first day of estrus) and continued every other day for 61 days post-estrus. Bitches in Group (B) were similarly injected with equal volumes of 0.9% saline. Blood samples, obtained prior to laparotomy and every other day for 85 days thereafter, were assayed for plasma progesterone concentrations using radioimmunoassay. One bitch in each of Groups (A) and (D) did not form luteal tissue following treatment with PMSG and HCG although both bitches exhibited estrus following treatment. All other bitches showed an increase in progesterone levels (4 to 19 ng/ml) between the first day of estrus and 10 days post-estrus. Thereafter, progesterone levels progressively declined in all groups with levels below 1 ng/ml between 38 to 40 days post-estrus. Results of this study suggested that CL formed in the bitch following PMSG and HCG treatment have a reduced function compared to non-induced CL of a normal, non-fertile estrous cycle. Such premature CL regression appears to be independent of the presence or absence of the uterus.

Effect of phenylbutazone (PBZ) on luteolysis in the mare induced by uterine biopsy

Uterine biopsy in the mare on day 4 post-ovulation causes an acute inflammatory reaction which results in premature luteolysis. In this study, seven mares (4 to 6 years of age) were used in a switchback experimental design to test the hypothesis that in the mare parenterally administered PBZ will block luteolysis induced by uterine biopsy on day 4 post-ovulation. All mares were allowed two normal estrous cycles (range 18 to 24 days). On the first day of estrus of the third estrous cycle each mare was intravenously given 2 grams PBZ (treatment) or 10 ml 0.9% saline (control) daily until signs of estrus were exhibited. The day of ovulation (day 0) was determined by rectal palpation and subsequently verified by peripheral plasma progesterone concentrations. On day 4 following ovulation all mares were subjected to uterine biopsy, and subsequent estrus detection was performed daily using an andro-genized gelding. A total of 19 estrous cycles (ten for PBZ treatment and nine for controls) were evaluated. Mean number of days (+/-SE) from uterine biopsy to induced estrus was 5.00+/-0.16 for control cycles and was significantly different (P<0.025) when compared with 9.20+/-0.34 days for treatment cycles. Results of this study suggest that PBZ can block luteolysis in the mare induced by uterine biopsy on day 4 post-ovulation, possibly as a result of accumulating PBZ in acutely inflamed uterine tissue and inhibiting prostaglandin synthesis.

A histological study of the effect of saline and povidone-iodine infusions on the equine endometrium

Abstract A study was conducted to assess the reaction of the endometrium of the mare to both saline and povidone-iodine infusions. In the control group (Group 1), uterine biopsies were taken at 0, 3, 5, 7, 10, 15, 20 and 30 days from the beginning of the experimental period. The treatment groups had intrauterine infusions of saline (Group 2) or 1% povidone iodine in saline (Group 3) on Days 0 and 2, and had endometrial biopsies taken on the same days as the control group. The concentration of inflammatory cells in the endometria of the Group-2 mares paralleled that of the Group-1 mares but was at a slightly higher level. Group-3 mares demonstrated significant increases in the numbers of inflammatory cells. An acute reaction was observed in Group-3 mares until Days 7 to 10. Thereafter, the inflammatory reaction changed in nature from an acute to a more chronic reaction. By Days 15 to 30, Group 3 still demonstrated increased signs of inflammation, including infiltration with eosinophils. The results of this study indicate that intrauterine infusion of 1% povidone-iodine solution in mares can cause chronic inflammatory changes in the endometrium.

Inability of progestogen pretreatment to prevent premature luteolysis of induced corpora lutea in the anestrous bitch

Fourteen mature anestrous bitches were used to determine the effectiveness of pretreatment with an orally active progestogen to prevent premature luteolysis of induced corpora lutea (CL) in the anestrous bitch. In Group 1, seven bitches were treated orally with megestrol acetate (Ovaban((R))) at the rate of 2.2 mg/kg body weight for eight days. Three days later, the bitches were treated daily with pregnant mares serum gonadotropin (PMSG) (44 IU/kg body weight) administered intramuscularly for nine consecutive days, and each bitch was given 500 IU human chorionic gonadotropin (HCG) on day 10, or on the first day of induced estrus if the bitches exhibited estrus while being treated with PMSG. A control group (Group 2) of seven bitches was not treated with Ovaban((R)) but was similarly given PMSG and HCG. Estrus was detected twice daily using a vasectomized male dog and verified by vaginal cytology. Blood samples were obtained on the first day of induced estrus (day 0) and every other day until day 90 post-estrus. Plasma progesterone (P(4)) concentrations were determined by a non-extraction solid phase radioimmunoassay (RIA), and data were analyzed by Students t-test. There was no significant difference between the progesterone profiles of both groups of bitches. In addition, P(4) values were less than 1 ng/ml by day 50 post-estrus. Results of this study suggested that pretreatment with an orally active progestogen was not effective in preventing premature luteolysis of induced CL in the anestrous bitch.

Ultrastructural Morphology of Plasma Cells in Normal Ovine Hemal Lymph Nodes

The fine structure of plasma cells from the hemal lymph nodes of five healthy sheep was examined. The plasma cells showed many cytoplasmic processes, indicating motility and phagocytic activity. They were randomly located in the medullary cords of the hemal lymph nodes. Their perinuclear spaces were irregular and contained granular material similar in density to that of rough endoplasmic reticulum. Some of the plasma cells provided morphological evidence of erythrophagocytosis at different levels. Such a phenomenon was previously reported only in pathological conditions. The red blood cell being engulfed by the plasma cell appeared smaller than normal. Most of the examined plasma cells showed Russells Bodies inside the cisternae of the rough endoplasmic reticulum of the healthy hemal lymph nodes. Some of these Russells Bodies had fuzzy borders. They were separated from the rough endoplasmic reticulum by evenly spaced halos.

A light and electron microscopic study of the periparturient bovine corpus luteum

Corpora lutea were obtained surgically from fifteen mature Angus crossbred cows representing three experimental groups of five cows each. Cows in Group A were 180 days of gestation, cows in Group B had recently experienced parturition (<or=24 hours) and cows in Group C had recently exhibited standing estrus (<or=24 hours). Samples of corpora lutea were fixed in 10% buffered formalin and prepared for light microscopy and 2.5% glutaraldehyde and processed for electron microscopy. Luteal cells from cows in Group A were of two distinct types, large cell and small cell types. The large luteal cells of both Groups A and B were similar, with one exception. Those from cows in Group B stained less intensely and the nuclear chromatin appeared less pronounced and marginated. Small luteal cells were not readily observed in corpora lutea from Group B. In addition, there was evidence of pyknosis, karyorrhexis and mononuclear cell infiltration in corpora lutea from Group B. Luteal cells from cows in Group C were characterized by atrophy of the nucleus and vacuolation of the cytoplasm. Vascular changes consisted of endothelial cell necrosis and pyknosis of cells of the tunica media of arteries within the corpus luteum. Ultrastructurally, there were distinct differences in all groups with respect to the size and quantity of lipid droplets, the presence of electron lucent vesicles in the cytoplasm and indentations of the nuclear membrane of luteal cells. The results of this study suggest that at the time of parturition in the cow luteolysis may involve primarily small luteal cells, and that the corpus luteum may be a source of progesterone, which is likely a function of the large luteal cells.