Alfred R. Menino

Characterization of porcine oocyte zonae pellucidae by polyacrylamide gel electrophoresis.

Summary Polyacrylamide gel electrophoresis of zonae pellucidae isolated from porcine ovarian oocytes and dissolved in either 6 M urea or 2.0% SDS suggest that the zona pellucida is composed of a single protein in addition to a carbohydrate component. The structural organization of the protein in the porcine zona pellucida is postulated to consist of repeating units of this single protein maintained by noncovalent bonding. Electrophoresis in SDS-β-MSH polyacrylamide gels has shown the protein to be composed of four polypeptide or protein subunits with molecular weights of 17,000 D, 57,000 D, 66,000 D and 85,000 D. These proteins may be cross-linked by disulfide bridges comprising the single protein visualized in gels without β-MSH. The subunit proteins may be in a multimeric arrangement supported by covalent bonds which maintain the complex structure. Relative percentages of the subunit proteins determined by scanning densitometry were: 50.3% (17,000 D), 23.7% (57,000 D), 18.6% (66,000 D) and 6.% (85,000 D). The unequal percentages reported by densitometry may be an indicator of the number of molecular subunit species within a complex.

Protein content and volume of early porcine blastocysts

Mean protein and volume of 222 blastocysts collected on 6 to 9 days of pregnancy were measured. Embryo protein differed (P < 0.05) for each day of development studied. Protein content of embryos doubled between days 6 and 7 and days 7 and 8 (1.2 ± 0.04, 2.0 ± 0.14, and 3.7 ± 0.2 μg, respectively). A dramatic increase from 3.7 ± 0.2 to 56.0 ± 3.4 μg was observed between days 8 and 9. Blastocyst volume increased (P < 0.05) from 0.56 ± 0.03 × 10−2mm3 to 1.11 ± 0.04 × 10−2mm3 between days 6 and 7, and then increased 10-fold on day 8 and five-fold on day 9. Blastocyst volume was not correlated with protein for days of development and females studied. Approximately 20% of all blastocysts within a single female contained less protein than the average protein content of all embryos from the same uterus. The results indicate that day 6 of development marks the onset of an exponential increase in embryo protein. Also, blastocyst volume is not correlated with blastocyst protein, suggesting that embryo viability is difficult to estimate by size alone. Further, approximately 20% of the blastocysts collected from a single female may exhibit reduced viability, based on reduced protein content, as early as day 6 of development.

Protein content of porcine embryos during the first nine days of development

Little is known about the physiochemical aspects of porcine embryos prior to implantation. The purpose of this study was to quantitate the total protein content of porcine embryos from fertilization through day 9 of development. Thirty-seven gilts and two sows were hand mated and the reproductive tracts collected at slaughter 1 to 9 days after the onset of estrus. The uteri were flushed with phosphate buffered saline (PBS), and embryos were collected, washed with PBS and transferred directly to test tubes containing distilled water. Only embryos which appeared morphologically normal were used. Protein content was determined by the Bio-Rad microassay. Standard curves were constructed for each assay using. 8 to 19 microg gamma globulin (Bio-Rad assay). Protein standards were run in triplicate. Regression lines were calculated for protein standards, and the resulting line was used to determine total protein in the unknown samples. Protein content of unfertilized eggs and embryos increased steadily through days 3, 4 and 5 of development, from 273 to 334, 491 and 620 ng, respectively. This result suggests that the protein content of porcine embryos increases only slightly from fertilization through day 5 of development. A dramatic increase in embryo protein content was observed between days 6 and 9 of development, which is the time of blastocyst formation and hatching of the embryo from the zona pellucida.

The influence of ovulation number and ovarian dimensions on embryo production in superovulated cattle

Abstract Twenty-one cycling Angus heifers and five Holstein cows received a subcutaneous (SC) injection of 50 mg of progesterone (P) in oil for 14 consecutive days. On day 6 of (P) treatment, animals were injected intramuscularly (IM) with 6 mg of estradiol valerate, and on day 13, received an IM injection of 2,000 IU of Pregnant Mare Serum Gonadotropin. Three additional Angus heifers were used as non-hormone treated controls. Seventeen of 21 heifers and 4 of 5 cows (81%) exhibited estrus within 48 to 132 hr following P treatment. Two of the five animals in which estrus was not observed were palpated as pregnant and discarded from the study. Treatment animals showing estrus were randomly assigned either to Group I, animals bred by natural service, or Group II, animals artificially inseminated with two straws of frozen semen at 12-hr intervals for a total of four breedings. Twenty-one animals were slaughtered 2 to 6 days after the onset of estrus, and those animals in which estrus was not detected were slaughtered 10 days after the last P injection. Two of the 24 treated animals had no ovulations. A total of 397 ovulation points ( 397 22 ) were counted for a mean ovulation rate of 18 ovulations per animal. One hundred and fifty-six ova were recovered ( 156 397 ) for a collection rate of 39%. Group I animals had 44 of 66 (67%) of their ova fertilized while 23 of 71 (32%) of the ova in Group II were fertilized. Nineteen unfertilized eggs were collected from the three animals not observed in estrus. No differences in fertilization rates between the Group I and Group II animals were found. Mean ovarian width, length and weight in the treated animals was measured and found to be 3.5 ± 1.1 cm, 4.8 ± 1.4 cm, and 21.7 ± 21.2 gm, respectively. Ovarian width, length and weight were all positively correlated with the number of ovulations per ovary r=.74, r=.74, and r=.55, respectively. No significant correlation existed between ovarian width (r=.16), lenght (r=.21), or weight (r=.13) when compared to ova recovery rate. This result suggests that ovarian size or weight may not be the limiting factor involved in embryo recovery.

Superovulation, fertilization and embryo recovery in gonadotropin treated prepubertal calves

Abstract Prepubertal Angus calves between 4 and 7 months of age, weighing 135 to 180 kg, were randomly assigned to one of four superovulation treatments with seven calves per treatment group. Group I calves were injected intramuscularly (im) with 1200 I.U. of pregnant mares serum gonadotropin (PMSG). Group II calves were injected with 1200 I.U. PMSG followed in 72 hrs with 50 mg luteinizing hormone (LH) given intravenously (iv). Group III calves were injected (im) twice daily for five days with 5, 4, 3, 2, and 1 mg of follicle stimulating hormone (FSH) respectively, for a total of 30 mg of FSH. Group IV calves were given 30 mg FSH, as in Group III, followed by 50 mg LH (iv) 96 hrs after the first FSH injection. All calves were inseminated by the rectal fixation method with multiple doses of frozen semen. Embryos were recovered surgically or after slaughter 48 to 72 hrs following the last insemination. The ovulation rate was 2.0, 11.3, 2.0 and 21.9 for Groups I, II, III and IV, respectively. Recovery and fertilization rates were 1.1 and 0.3, 8.0 and 5.6, 1.4 and 0.3, and 10.9 and 3.6 embryos for Groups I–IV, respectively. In this study, treatment with LH increased the ovulation, fertilization and embryo recovery rates in calves treated with PMSG or FSH.

Development of one-cell porcine embryos in two culture systems.

The effect of culture system and protein supplementation on the development of one-cell porcine embryos was investigated. One hundred and sixty-four one-cell embryos were collected at slaughter from 19 handmated gifts. Culture systems evaluated were microdrops of Whittens medium (WM) under paraffin oil (M) or tubes with 4.0 ml of WM (T). Also tested was T with a 2.0 ml paraffin oil overlay (O). Media evaluated were WM with: no bovine serum albumin (-BSA), 15 mg/ml BSA (+BSA) or 10% (v/v) heat-treated porcine serum (+HTPS). Twenty-nine, 29, 28, 29, 28 and 21 one-cell embryos were assigned to treatments T + BSA + O, T - BSA, M + BSA, M - BSA and M + HTPS, respectively. All cultures were conducted at 37 C in a humidified atmosphere of 5% CO2 in air. At the cessation of division, embryos were fixed and stained, and each was assigned a cleavage index bases on the number of divisions completely in culture. The mean cleavate indexes for embryos cultured in T + BSA + O (2.07) and T + BSA (1.97) were not significantly different, but both were greater (P less than .05) than the indexes for T - BSA (1.09), M + BSA (1.12), M - BSA (.82) and M + HTPS (.86). Mean cleavage indexes for embryos cultured in T - BSA, M + BSA, M - BSA and M + HTPS were not significantly different. More (P less than .05) embryos developed to the blastocyst stage in T + BSA + O (6/29) than in either T - BSa (0.28) or M BSA (0.28). Fragmentation was greater (P less than .05) in embryos cultured in M - BSA than in embryos cultured in tubes. These results suggest that culture tubes of WM with 1.5% BSA are superior to microdrops and WM with 10% HTPS in supporting the in vitro development of one-cell porcine embryos.

Occurrence of a non-cellular suprazonary layer in embryos collected from prepuberal calves

Abstract Twenty Angus calves between 4 and 7 months of age were randomly assigned to one of two superovulation treatment groups. Group I consisted of ten calves which were injected intramuscularly with 50 mg of progesterone 4 and 2 days before injection with 1200 I.U. of PMSG followed 72 hrs later by 50 mg of LH given intravenously. Group II consisted of ten calves which were not injected with progesterone before receiving PMSG and LH as in Group I. Both groups of calves were inseminated by the rectal fixation method with two straws of frozen semen 72 hrs after PMSG injection and at subsequent 12 hr intervals for a total of four inseminations. All semen was extended from a single ejaculate from one bull. Embryos were recovered by surgery or slaughter 48 to 72 hrs following the last insemination. A total of 80 and 70 ovulations were recorded from treatment Group I and II, respectively. Recovery and fertilization rates were 66 and 57% following progesterone treatment and 67 and 51% in calves without progesterone pretreatment. Seventy-seven percent ( 23 30 ) of the fertilized embryos recovered in treatment Group I exhibited a suprazonary layer. This suprazonary layer appeared to be non-cellular on the basis of eosin-hematoxylin staining and ranged in thickness from 3 to 24 μm. All fertilized embryos in treatment Group II and unfertilized eggs in treatment Groups I and II, possessed completely smooth zonae pellucidae with no evidence of a suprazonary layer. These observations suggest that the conditions of the progesterone treated prepuberal tract, coupled with the process of sperm penetration of the oocyte, result in the formation of a non-cellular layer which surrounds the zona pellucida to varying degrees of thickness. The influence of this suprazonary layer on embryo viability in prepuberal calves remains to be determined.