T.C. Cantley

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor.

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.

Presence of β-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization

The present study examined the effect of β-mercaptoethanol (BME) during in vitro maturation (IVM) of pig oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration, subsequent embryo development and blastocyst cell numbers. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU)-23 medium containing porcine follicular fluid, cysteine, hormonal supplements and 0 to 50 μM BME for 20 to 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20 to 22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5 to 6 h. Putative embryos were transferred to NCSU-23 containing 0.4% BSA and cultured for 144 h (Experiment 1). In comparisons between the presence or absence of BME, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, compared with no addition (26%), the presence of 12.5 and 25 μM BME increased (P<0.05) the proportion of blastocysts in a dose-dependent manner (34 and 41%). Further increase from 25 to 50 μM BME reduced (P<0.05) the blastocyst development rate. Blastocysts derived from oocytes matured with 25 μM BME had the highest (P<0.05) trophectoderm (TE) and total cell numbers. No difference was found in inner cell mass (ICM) cells among treatments. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.01) level of GSH was found in oocytes matured with 25 μM BME. Compared with 25 μM BME, GSH was low (P<0.05) at 50 μM BME. The results show that at certain concentrations BME in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in pig oocytes.

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.

Effects of Culture Medium, Serum Type, and Various Concentrations of Follicle-Stimulating Hormone on Porcine Preantral Follicular Development and Antrum Formation In Vitro

Abstract Developing a culture system for preantral follicles has important biotechnological implications due to the potential to produce large number of oocytes for embryo production and transfer. As an initial step toward accomplishing this long-term goal, a study was conducted to determine the effects of culture medium, serum type, and different concentrations of FSH on preantral follicular development in vitro. Specific endpoints included follicular growth rate, antrum formation, recovery rate of cumulus cell-oocyte complexes (COCs) from follicles, and oocyte meiotic competence. Compared with the North Carolina State University medium 23 (NCSU23), preantral follicles cultured in TCM199 medium for 4 days grew faster (P < 0.02). However, more follicles cultured in NCSU23 differentiated to form an antrum than in TCM199 (P < 0.01). For this reason, NCSU23 was chosen to investigate the role of FSH and serum type in regulating preantral follicular growth. Compared with the 0 mIU/ml FSH control, addition of 2 mIU/ml FSH to the medium stimulated follicular growth and antrum formation and suppressed apoptosis of granulosa cells (P < 0.05), supporting the essential role of FSH in preantral follicular growth and development. Another experiment compared fetal calf serum (FCS) with prepubertal gilt serum (PGS) and studied different concentrations of FSH in the culture medium (0.5, 1, and 2 mIU/ml). The best follicular growth rate was obtained with 2 mIU/ml compared with 0.5 or 1 mIU/ml FSH. Compared with PGS, FCS supplementation increased the cumulative percentage of antral follicles and COC recovery rate (P < 0.04). None of the oocytes recovered from any of these experiments reached metaphase II stage after maturation in vitro. In summary, culture medium, serum type, and FSH concentration in the medium interacted to affect follicular growth and antrum formation in vitro. These results suggest that a longer term culture of preantral follicles (>4 days) may be needed to produce oocytes capable of undergoing meiosis in vitro.

Successful nonsurgical deep uterine embryo transfer in pigs

At present, it is possible to transfer pig embryos directly into the uterine body of sows by nonsurgical procedures. The aim of this study was to develop a procedure for nonsurgical embryo transfer (ET) into the upper part of one uterine horn in gilts and sows. In experiment 1, 29 gilts and 43 sows were used. Intrauterine insertions took place for each female at days 4-6 of the estrous cycle (D0 = onset of estrus). An artificial insemination (AI) spirette was inserted into the cervix to assist with the guidance of a modified flexible catheter originally developed for deep intrauterine insemination in pigs. The flexible catheter length inserted anterior to the inserted AI spirette was 43.0 +/- 1.7 cm. The time required to complete the procedure was affected by the type of female (P < 0.001) and by the difficulties encountered for inserting the catheter (P < 0.001). However, when no or minor difficulties were encountered during the insertion of the catheter (in approximately 70 and 80% of gilts and sows, respectively), the time required to complete the procedure did not differ between gilts (2.5 +/- 0.1 min) and sows (2.3 +/- 0.1 min). In experiment 2, 24 to 31 fresh morulae and/or blastocysts were transferred to each of 24 recipients. Seventeen animals (70.8%) farrowed an average of 6.9 +/- 0.7 piglets, of which 0.6 +/- 0.3 piglets were born dead. In conclusion, the procedure described in this study offers new possibilities to transfer embryos nonsurgically to the uterine horn of pigs.

Hormone response of dairy cows with ovarian cysts after treatment with HCG or GnRH

Abstract Twenty lactating Holstein and Guernsey cows, diagnosed by rectal palpation as having ovarian cysts, were randomly divided within breed into two groups to receive either a single intramuscular injection of 100 μg of synthetic gonadotropin releasing hormone (GnRH) or an intravenous injection of 10,000 IU of human chorionic gonadotropin (HCG). The objective was to compare hormonal and clinical changes in cows with ovarian cysts following treatment with GnRH and HCG. Eight of ten and nine of ten cows given either GnRH and HCG, respectively, responded to treatment and subsequent fertility was not different between the two groups. Pre-injection plasma levels of LH, progesterone, and estradiol were highly variable. Mean plasma levels of LH, progesterone and estradiol did not differ between groups either following treatment (days 1–17 post-treatment), at the subsequent estrus, or during days 1–13 following the subsequent estrus. Mean LH levels did not differ significantly on the days either post-treatment or post-estrus except that levels were higher (P

Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro

The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

Developmental ability of porcine oocytes matured and fertilized in vitro.

The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whittens medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42 71 ) and with monospermic penetration and male and female pronuclei (32%, 23 71 ) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18 71 and 8%, 6 71 , respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28 288 and 13%, 41 318 , respectively) were also higher (P < 0.05) than in controls (2%, 6 284 and 6%, 16 248 , respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9 288 in PFF-treated group and 2%, 6 284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3 100 ) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

Effect of time of ovulation and sperm concentration on fertilization rate in gilts

In normal production practices, sows and gilts are inseminated at least twice during estrus because the timing of ovulation is variable relative to the onset of estrus. The objective of this study was to determine if a normal fertilization rate could be achieved with a single insemination of low sperm number given at a precise interval relative to ovulation. Gilts (n=59) were randomly assigned to one of three treatment groups: low dose (LD; one insemination, 0.5 x 10(9) spermatozoa), high dose (HD; one insemination, 3 x 10(9) spermatozoa) or multiple dose (MD; two inseminations, 3 x 10(9) spermatozoa per insemination). Twice daily estrus detection (06:00 and 18:00 h) was performed using fenceline boar contact and backpressure testing. Transrectal ultrasonography was performed every 6 h beginning at the detection of the onset of standing estrus and continuing until ovulation. Gilts in the LD and HD groups were inseminated 22 h after detection of estrus; MD gilts received inseminations at 10 and 22 h after detection of estrus. Inseminations were administered by using an insemination catheter and semen was deposited into the cervix. The uterus was flushed on Day 5 after the onset of estrus and the number of corpora lutea, oocytes, and embryos were counted. Time of insemination relative to ovulation was designated as 40 to >24 h, 24 to >12 h, and 12 to 0 h before ovulation and >0 h after ovulation. The LD gilts had fewer embryos (P<0.04), more unfertilized oocytes (P<0.05) and a lower fertilization rate (P<0.07) compared to MD gilts. The effects of time of insemination relative to ovulation and the treatment by time interaction were not significant. We conclude that a cervical insemination with low spermatozoa concentration may not result in acceptable fertility even when precisely timed relative to ovulation.

The effects of zearalenone on reproduction in swine. II. The effect on puberty attainment and postweaning rebreeding performance

Eighty-five prepuberal, crossbred gilts received, ad libitum, a diet containing 0 or 10 ppm purified zearalenone for 30 d beginning at 145 to 193 d of age. At the end of this period all gilts were placed on the control diet and exposed daily to a mature boar for 60 d. Within 3 to 5 d of zearalenone ingestion, gilts showed marked vulval swelling and reddening, which continued for the 30-d feeding period. Thereafter symptoms slowly subsided. Zearalenone treated gilts showed first estrus significantly later than controls (P<0.05), but the proportion of gilts showing estrus within 60 d of boar exposure was similar (P>0.05). The length of the first estrous cycle was not affected by the ingestion of zearalenone before puberty (P>0.05). In a second study, 65 multiparous, crossbred sows were full-fed twice daily a ration containing 0 or 10 ppm of purified zearalenone beginning 14 d before weaning. Postweaning, all sows were fed the control diet, were checked for estrus daily, and inseminated at the first postweaning estrus. Neither sows nor gilts from their litters exhibited signs of hyperestrogenism during treatment. Weaning to estrus interval was significantly extended in zearalenone treated sows (P<0.05), but all other variables of fertility assessed were similar. These data suggest that zearalenone ingestion before puberty delays the stimulation of puberty associated with boar exposure, but does not affect subsequent cyclicity if zearalenone is removed from the ration. Similarly, zearalenone ingestion during lactation delays the return to estrus after weaning, but does not affect subsequent fertility when removed from the ration at weaning.