Instructing the animal physiology graduate student in human assisted reproductive technology.

Animal physiology graduate students provide an excellent personnel resource for laboratories performing human assisted reproductive technology (ART) procedures. However, the basic training of these students falls short of what is required for this highly specialized field. We designed a course to enhance their education in this area via classroom and hands-on laboratory instruction in a hospital and university setting. Topics covered in the course included in vitro maturation, in vitro fertilization, embryo culture, embryo transfer, quality control, quality assurance, micromanipulation, and cryopreservation. These techniques were applied to a group project to evaluate the influence of spermatozoal quality and quantity on early embryonic development in cattle and humans. Student grades were based on 1) oral and written examinations; 2) demonstrated competency in laboratory techniques; 3) presentation of class project data at a state academy of science meeting; and 4) initiative, determination, and interest in the coursework. Three aspects of the course stood out as very positive. First, the team approach to accomplishing a class project was new to some of the graduate students. Second, a bond was formed between hospital- and university-based faculty that did and will continue to foster unique teaching and research opportunities between the two groups. Third, the opportunity for students to present research data in a formal setting was very rewarding. This course made the students keenly aware of the many aspects of ART and provided them with specialized skills that should make them more marketable in the field of reproductive technology.

Oviductal fluid and growth factors failed to enhance development of porcine embryos.

Porcine embryos (1-, 2- and 4-cell) were cultured in a basal medium consisting of Krebs-Ringer bicarbonate buffer supplemented with oviductal fluid and several growth factors and observed for further development. Oviducts were flushed at either 48 h (Experiment 1) or 96 h (Experiment 2) after the onset of estrus. Observations were made every 48 h (Experiment 1) or 12 h (Experiment 2) until failure of the embryos to develop for 2 consecutive observations. Embryos were scored 0 = no development, 1 = cleavage, 2 = morula, 3 = blastocyst, or 4 = hatched blastocyst. In the first experiment, development of 1-, 2- and 4-cell embryos (n=282) in the basal medium supplemented with oviductal fluid (4:1) or 3 sets of growth factors, was less or equal to one cleavage stage. Those embryos cultured in the basal medium supplemented with bovine serum albumin (fatty acid free) (BSA) advanced to the blastocyst stage. In the second experiment, 96 h aged embryos (n=142) were cultured in the basal medium supplemented with IGF-1 and - 2 and EGF, or with BSA alone or with BSA and the three growth factors. In the treatments without BSA, the embryonic development was less than one cleavage, whereas in those treatments with BSA, embryos advanced beyond hatching and began to expand. We conclude that for culture of porcine embryos, supplementation with several growth factors or with oviductal fluid, in the concentration used in this study, was of little benefit at this stage of development. However, the type of BSA significantly affected development. More than 90% of the embryos reached the morula and blastocyst stages in medium than included BSA (fatty acid free).

Stimulation of development of in vitro-matured and in vitro-fertilized bovine embryos by platelets.

In vitro-fertilized bovine embryos were incubated in Menezo's B2 medium (MB2) supplemented with 2 mg/mL of BSA. In Exp. 1, eight-cell stage embryos were allotted to one of the following groups: control medium (MB2), MB2 with 20 ng/mL of platelet-activating factor (PAF), 1 x 10(7) bovine blood platelets (Platelets), oviductal cells (BOEC), BOEC and 20 ng/mL of PAF (BOEC+PAF), or BOEC and 1 x 10(7) platelets (BOEC+Platelets). In Exp. 2, eight-cell embryos were allotted to one of the following groups: control medium (MB2), MB2 with 1 x 10(7) platelets (Platelets), 1 x 10(7) platelets and 10 micrograms/mL of platelet-derived growth factor antibody (Platelets+anti-PDGF), 1 x 10(7) platelets and 1 microgram/mL of indomethacin (Platelets+Indomethacin), or 1 x 10(7) platelets and 3 micrograms/mL of mianserin (Platelets+Mianserin). Embryos were incubated at 39 degrees C in 5% CO2 in groups of five until 8 d after in vitro fertilization (IVF). In Exp. 1, Platelets stimulated embryo development to the morula, blastocyst, and expanded blastocyst stages. Embryo development was greatest in the BOEC+Platelets group on d 7 and 8 after IVF. Only embryos incubated in the BOEC+Platelets treatment group reached the hatched blastocyst stage on d 8. In Exp. 2, embryos incubated in the Platelets treatment group had the greatest (P < .05) proportion develop beyond the eight-cell stage. Embryos incubated in the Platelets + anti-PDGF group had less (P < .05) development beyond the eight-cell stage and to the morula stage. These results indicate that the stimulatory effects of PDGF on bovine embryo development may be derived from both the oviductal epithelium and platelets.

Culture of ovine embryos in the absence of bovine serum albumin.

Bovine serum albumin (BSA), a relatively impure protein, is routinely used as a component of embryo culture media. Since media containing BSA are chemically undefined, it would be desirable to replace BSA with substitutes of similar activity which are either chemically better defined and/or better standardized than BSA. Two commercial products, Ultroser((R)) G (USG) and Solcoseryl((R)) (SOL), were evaluated as replacements for BSA in culture with respect to the development of ovine embryos in vitro. A total of 126 late 8-cell and early 16-cell embryos were distributed among modified Brinster's medium for ovum culture (BMOC-2) containing either 1.5% BSA, 2.0% USG or 2.0% SOL. All three culture media supported development of ovine embryos. Results indicate that 8- and 16-cell embryos will develop into blastocysts in a BSA-free medium containing either USG or SOL. A higher number of embryos developed into blastocysts in media containing BSA than in media containing USG or SOL, and more blastocysts hatched in media containing BSA. Although the overall degree of embryonic development was more advanced in BSA-supplemented media, the concentrations of USG and SOL that were used in this study may not have been optimal for ovine embryo culture.

Gross and histological characterization of endometrial tissues from SLA miniature pigs with cystic endometrial hyperplasia.

Cystic endometrial hyperplasia (CEH) is a uterine disorder characterized by the formation of large numbers of cysts in the endometrium. The purpose of this study was to examine and characterize cell types in the endometrium associated with the cysts and uterine glands. No apparent histological differences between CEH-involved and normal uterine columnar epithelium were found. Endometrial glands in CEH-involved and normal uteri were lined with simple or ciliated columnar epithelial cells and surrounded by lamellar connective tissue. The cyst epithelium appeared to be stretched obliquely and compressed so that both the cells and nuclei were horizontally oriented relative to the cyst lumen and were surrounded by lamellar connective tissue. Electron microgaphs revealed an abnormally high number of mitochondria in the cystic cells as compared to normal glandular cells. In conclusion, CEH is characterized by the formation of cysts which develop from the uterine glandular tissue. Epithelial cells lining the glands appeared to be distorted, possibly in response to internal pressure from increased volume due to high metabolic activity, and/or no uterine luminal opening.

Effects of unilateral castration and unilateral cryptorchidism of the Holstein bull on plasma gonadotropins, testosterone and testis anatomy.

The effects of unilateral castration (UC) and induced unilateral cryptorchidism (CR) on plasma hormones and testis anatomy were studied in 36 Holstein bulls altered at either 3, 6 or 9 mo of age (n = 12). Plasma hormone concentrations were determined in six samples collected at hourly intervals on d 0, 1, 3, 7, 14 and 30, and then at monthly intervals through 6 mo after gonadal manipulation. Although plasma testosterone (T) showed a transient decrease (P less than .05) immediately after treatment, mean plasma concentrations of luteinizing hormone (LH) and T were unaffected by UC or CR over the 6-mo period (P greater than .05). Both hormones increased (P less than .05) in concentration with advancing age. Plasma follicle stimulating hormone (FSH) concentration was greater (P less than .05) in UC than in intact (IN) bulls overall, while FSH in CR bulls did not differ (P greater than .05) from either group. At slaughter, 11 mo after gonadal alteration, mean testis weight, ratio of testis weight to body weight and mean testis sperm cell numbers were increased (P less than .05) in UC bulls compared with mean testis values in intact (IN) bulls. Unilateral castration increased (P less than .05) seminiferous tubuler diameter and seminiferous epithelial cell height from basement membrane to the border of the lumen, but did not alter the ratio of tubuler to interstitial space within the testis. Seminiferous tubuler diameter and epithelial cell height were increased (P less than .05) in CR compared with IN bulls. Unilateral gonadal alteration at 3 mo of age caused a greater (P less than .05) hypertrophy of the scrotal testis in both UC and CR bulls than alteration at 6 or 9 mo of age. Results indicate that unilateral gonadal disruption is followed by rapid compensation in testis T production, little change in systemic LH and a rapid increase in secretion of FSH in the bull within those ages investigated. Further, UC elicits a greater compensatory hypertrophy than CR and the pituitary-testis endocrine axis is more responsive to alteration at 3 mo than at 6 or 9 mo of age in the bull.

Effects of unilateral castration and unilateral cryptorchidism of the Holstein bull on in vitro Leydig cell response.

The effects of unilateral castration (UC) and induced unilateral cryptorchidism (CR) on in vitro Leydig cell function were determined utilizing 36 Holstein bulls altered at either 3, 6 or 9 mo of age. Testes were removed 11 mo after gonadal manipulation and Leydig cells were dispersed in media containing 0 or 75 ng luteinizing hormone (LH). After incubation for 4 h, testosterone (T) concentration in the media was determined by radioimmunoassay. Leydig cells of UC animals produced greater (P less than .001) amounts of T than did Leydig cells of either CR or intact (IN) bulls with either 0 or 75 ng LH. Leydig cell T response was greater (P less than .001) in UC animals altered at 3 mo of age than in those altered at 6 or 9 mo of age. In a second experiment using only UC bulls altered at 3 mo of age, similar results were obtained. Leydig cells of UC bulls produced greater (P less than .05) amounts of T in vitro, both without LH or in response to 75 ng LH, than did Leydig cells of IN bulls at 10 mo after gonadal manipulation. Results indicate that UC in the bull causes increased Leydig cell capacity for T production in the remaining hypertrophied testis and this effect is greater when UC is performed at 3 mo of age than at 6 or 9 mo of age.

Testosterone, luteinizing hormone, follicle stimulating hormone, and prolactin response to unilateral castration in prepubertal Holstein bulls.

Hemicastration of Holstein bulls at 3 months of age resulted in increased (P<0.005) testicular weitht and testis sperm cell content at 330 days after treatment, but did not alter sperm cell concentration in the remaining hypertrophied testis. Radioimmuroassay of blood hormones at 1, 6, 12, and 24 weeks after treatment revealed that unilateral castration did not alter (P>0.1) basal levels or GnRH response profiles of either LH or testosterone compared to intact bulls. Hemicastration caused FSH to be elevated (P<0.01) compared to intact bulls at all sampling periods in both unstimulated and GnRH stimulated bulls. Prolactin varied with season and was greater (P<0.001) in hemicastrated bulls than in intact bulls at 1 and 6 weeks after treatment. Results indicate that unilateral castration at 3 months of age caused testicular hypertrophy of both steroidogenic and gametogenic function and this phenomena may be triggered by increased FSH or prolactin secretion, or both. Further, results indicate different testicular regulation mechanisms exist for pituitary LH and FSH release in bulls.

In vitro mycotoxin binding to bovine uterine steroid hormone receptors.

The mycotoxins, aflatoxin B(1), aflatoxin M(1), aflatoxicol and zearalenone were tested for binding to bovine endometrial estrogen and progestin receptors. Radioinert estradiol-17beta, estrone, testosterone, and cholesterol were evaluated for binding to the estrogen receptor. Zearalenone and aflatoxicol but not aflatoxins B(1) and M(1) competed with estradiol-17beta for the estrogen receptor. The order of binding affinities for the estrogen receptor were zearalenone > estradiol-17beta > estrone > aflatoxicol. The affinity of zearalenone for the estrogen receptor was 2-3 times that of estradiol-17beta. Progesterone, cortisol, radioinert R 5020, and cholesterol were evaluated for binding to the progestin receptor. None of the tested compounds except R 5020 and progesterone competed for the progestin receptor. The significance of aflatoxicol binding to the estrogen receptor is unclear. It is proposed that aflatoxicol binding to the receptor may alter gene expression in target tissues or act at the level of the hypothalamus to inhibit gonadotropin secretion and ovulation. These effects could explain reports of reduced fertility in domestic animals following ingestion of aflatoxin contaminated feedstuffs. It is also suggested that the mechanism of adverse effects on fertility of chronic aflatoxin ingestion in cattle and other livestock should be more thoroughly investigated.

Endogenous hormone response and fertility in dairy heifers after treatment with Lutalyse and GnRH.

Fifty-five dairy heifers were given two injections of Lutalyse 11 days apart. Twenty-one of the heifers were also given an injection of GnRH 48 hr after the second Lutalyse injection (Group G). Of the remaining 34 animals, 19 were randomly allotted to be inseminated 12 hr after observed estrus following Lutalyse (Group E), while 15 were inseminated 80 hr after the second Lutalyse injection (Group P). The intervals from second Lutalyse injection to occurrence of both estrus and peak gonadotropin concentrations were variable among animals receiving only Lutalyse. GnRH injections reduced variation (P<.01) in the interval from second Lutalyse injection to occurrence of peak gonadotropin concentrations, but did not improve fertility. Pregnancy rates did not differ (P>.05) among treatment groups. The failure of GnRH administration following Lutalyse to improve pregnancy rates indicates that GnRH administration followed by insemination 12 hr later is not effective in increasing pregnancy rates above those attained in animals inseminated at either 12 hr post estrus or 80 hr after second Lutalyse injection.

Effect of unilateral castration and unilateral cryptorchidism on gonadotropin and testosterone response to gonadotropin releasing hormone in the bull.

The effects of unilateral castration (UC) and induced unilateral cryptorchidism (UCR) on basal plasma luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone, and on the responses of these hormones to gonadotropin releasing hormone (GnRH), were investigated in bulls altered at 3, 6 or 9 months of age. Blood plasma was collected before and after GnRH (200 micrograms) stimulation approximately 1 year following gonadal manipulation. Neither mean baseline concentrations nor GnRH-induced increases in plasma testosterone were altered (P greater than .1) by hemicastration or UCR (P greater than .1). Both mean baseline LH and GnRH-induced LH release were greater (P less than .05) in bulls altered at 3 months of age than in bulls altered at 9 months of age. UC increased (P less than .05) plasma LH response to GnRH over that observed in intact bulls, but not above that in UCR bulls. UCR had no detectable effect on either baseline concentrations or GnRH-stimulated LH release. FSH was increased (P less than .05) in hemicastrates, while UCR had a variable effect on peripheral FSH: FSH was reduced (P less than .05) in UCR animals altered at 3 months of age but increased (P less than .05) in UCR bulls altered at both 6 and 9 months of age when compared to FSH in intact bulls. The results indicate that, compared with intact bulls, UC bulls release increased amounts of both gonadotropins but similar amounts of testosterone in response to GnRH stimulation. UCR had a variable effect on FSH release and did not alter either LH or testosterone.

Tissue concentrations of mast cells and lymphocytes of the bovine uterine tube (oviduct) during the estrous cycle.

Estrous cycle fluctuations in the mast cell and lymphocyte counts (per mm2 of tissue) of the ampulla and isthmus of the bovine uterine tube (oviduct) were determined. Four random sections of each region of the uterine tube were prepared at 2 micron thickness and stained with toluidine blue. The mast cell count of the isthmus was found to be significantly higher (P less than 0.01) than the mast cell count of the ampulla for all stages of the estrous cycle. A significant increase (P less than 0.01) in the mast cell value of the isthmus occurred during metestrus, diestrus, and proestrus. In the ampulla, metestrous and diestrous mast cells numbers were significantly higher (P less than 0.05) than mast cell numbers during estrus. The lymphocyte numbers remained relatively constant in both the ampulla and the isthmus except during diestrus, when ampullary lymphocyte numbers increased significantly (P less than 0.05). Lymphocytes were observed to migrate through the uterine tube epithelium and mast cells were observed only in the lamina propria.

Influence of a progestogen on the cytophysiologic character of the bovine adenohypophysis.

The objective in the present experiment was to study the effects of melengestrol acetate (0.5 mg for 14 days starting at day 15 of the estrous cycle) on the cytophysiologic character of the bovine adenohypophysis. Twelve Hereford and Angus heifers were used. A combination of thick-thin sectioning of resin-embedded material was used to aid in specific identification of individual hypophyseal cells. Alterations were not observed in the cytophysiologic activity of the presumptive prolactin-, somatotropin-, thyrotropin- or corticotropin-secreting cells. A marked increase in chromophobes was observed in animals given melengestrol acetate. Evidence indicated that these chromophobes originated from gonadotropin-secreting cells.

Influence of a progestogen on carbohydrate histochemical and histologic features of the ampulla of the bovine uterine tube.

In the present experiment, study was made of the effects of melengestrol acetate given to heifers (0.5 mg for 14 days, starting at day 15 of the estrous cycle) on carbohydrate histochemical and histologic features of the ampulla of the uterine tube (oviduct). Melengestrol acetate-treated animals had significantly (P less than 0.5) fewer fertilized ova at 3 days after mating (50%) as compared with control animals (100%). Estrussynchronization treatment with melengestrol acetate reduced the amount of cytoplasmic and nuclear extrusions from the secretory cells of the uterine tubular ampulla, but did not alter the amount or types of mucosubstances within these cells.

PIP: Only heifers with estrous cycles of 18-22 days were used; 20 were in the experimental group and 20 were controls. Melengestrol acetate, .5 mg/animal/day for 14 days, was added to the grain ration, starting on Day 15 of an estrous cycle. Following drug withdrawal, each heifer was mated with 2 bulls at the synchronized estrus and killed 3 days later. Control heifers were killed after mating. The uterine tubes were flushed with saline solution to remove ova. An ovum was considered fertilized if cleavage had occurred and sperm were in the zona pellucida. Tubal tissues were prepared and studied with different stains. All heifers had ovulated. Melengestrol-acetate-treated heifers had signficantly (p less than .5) fewer fertilized ova 3 days after mating. Estrous synchronization treatment with melengestrol acetate reduced the amount of cytoplasmic and nuclear extrusions from the secretory cells of the tubular ampulla but did not alter the amount or types of mucosubstances within these cells. The major steroidogenic effect was considered to be estrogenic. However, the histologic and histochemical alterations observed were not estrogenic. Estrogen-dependent physiologic responses in reproductive tissues may be related to the number of estrogen-binding sites.