Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes

Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at -80 degrees C in groups of 10-20 (GSH) or 5-10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P < 0.05) of GSH (n = 207, 9.82 +/- 0.71 pmol/oocyte; n = 104, 9.73 +/- 0.81 pmol/oocyte; n = 108, 7.89 +/- 0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n = 217, 36.26 +/- 11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n = 70, 0.97 +/- 0.07 pmol/oocyte; TCM-PVA, n = 117, 0.81 +/- 0.13 pmol/oocyte; TCM-MAP, n = 107, 1.02 +/- 0.18 pmol/oocyte; NCSU-pFF, n = 134, 0.71 +/- 0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes.     

In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogroup

Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.     

Identification and content of insulin-like growth factors in porcine oviductal fluid.

Oviductal fluid (OvF) was collected from gilts by indwelling catheters during the estrous cycle and analyzed for content of insulin-like growth factors (IGF-I, IGF-II). Group 1, composed of 9 gilts in a summer environment near a boar, yielded mean daily fluid volumes of 1.18 +/- 0.16 ml during estrus and 0.69 +/- 0.03 ml post-estrus. Group 2, composed of 7 gilts in a moderate-temperature, light-regulated room, yielded 1.20 +/- 0.18 ml during estrus and then OvF flow essentially stopped. Serum samples were also collected 2 times daily during the cycle and analyzed along with the OvF for IGF-I and IGF-II. Serum was also analyzed for estradiol-17 beta (E2). For group 1, OvF content (concentration x fluid volume) of IGF-I and IGF-II was greater (p less than 0.05) at estrus than pre- and post-estrus. Daily mean content values (ng/day) for IGF-I and IGF-II during peri-estrus were 30.9 +/- 6.3 and 62.2 +/- 12.3, respectively. During nonestrus, values were 6.8 +/- 6.3 and 11.7 +/- 12.3, respectively. For group 2, OvF content of IGF-I and IGF-II during estrus was similar to that of group 1. Whereas IGF content differed between estrus and nonestrus periods, IGF concentrations were similar (p greater than 0.05), a finding that results from the difference in OvF produced. Compared with OvF concentrations of IGF for a given pig, blood plasma concentrations of IGF-I and IGF-II were 2- to 5-fold higher in the plasma sample collected the same day.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of prostaglandin F2alpha as the luteolysin in swine: III effects of estradiol valerate on prostaglandin F, progestins, estrone and estradiol concentrations in the utero-ovarian vein of nonpregnant gilts

Polyvinyl catheters were placed into the right and left utero-ovarian veins and saphenous vein and artery of three control (C) and four estradiol valerate (EV) treated gilts on Day 9 after onset of estrus. The EV treated gilts received 5mg EV/day on Days 11 through 15 after onset of estrus. On Days 12 through 17 utero-ovarian vein blood samples were collected at 15 min intervals from 0700 to 1000 hr and 1900 to 2200 hr and single samples were taken at 1100 and 2300 hr. Peripheral blood samples (saphenous vein or artery) were taken at 0700, 1100, 1900 and 2300 hr from Day 12 until the control gilts returned to estrus or until Day 25 for EV treated gilts and used to measure plasma steroid hormone concentrations. Utero-ovarian vein prostaglandin F (gf) concentrations (ng/ml, n-1,177) were measured by RIA. Status (control vs EV treated gilts) by day interactions were detected (P=.10). Curvilinear day trends were detected for plasma PGF concentrations in control (P less than .01) but not EV treated gilts. PGF concentrations (X +/- S.D.) for control and EV treated gilts were 1.20 +/- 2.08 and .26 +/- .84 ng/ml, respectively. PGF peaks (concentrations greater than X + 2 S.D.) occurred with greater frequency in control gilts (X2 =4.87; P less than .05). The interestrus interval (X +/- S.E.) for control and treated gilts was 19.0 +/- .6 and 146.5 +/- 74.8 days, respectively. Data indicate tht t estradiol valerate may exert its luteotrophic effect by preventing PGF release from the uterus.     

Influence of stage of the estrous cycle on endogenous opioid modulation of luteinizing hormone, prolactin, and cortisol secretion in the gilt

Fourteen gilts that had displayed one or more estrous cycles of 18-22 days (onset of estrus = Day 0) and four ovariectomized (OVX) gilts were treated with naloxone (NAL), an opiate antagonist, at 1 mg/kg body weight in saline i.v. Intact gilts were treated during either the luteal phase (L, Day 10-11; n = 7), early follicular phase (EF, Day 15-17; n = 3), or late follicular phase (LF, Day 18-19; n = 4) of the estrous cycle. Blood was collected at 15-min intervals for 2 h before and 4 h after NAL treatment. Serum luteinizing hormone (LH) concentrations for L gilts averaged 0.65 +/- 0.04 ng/ml during the pretreatment period and increased to an average of 1.3 +/- 0.1 ng/ml (p less than 0.05) during the first 60 min after NAL treatment. Serum prolactin (PRL) concentrations for L gilts averaged 4.8 +/- 0.2 ng/ml during the pretreatment period and increased to an average of 6.3 +/- 0.3 ng/ml (p less than 0.05) during the first 60 min after NAL treatment. Serum PRL concentrations averaged 8.6 +/- 0.7 ng/ml and 7.6 +/- 0.6 ng/ml in EF and LF gilts, respectively, prior to NAL treatment, and decreased (p less than 0.05) to an average of 4.1 +/- 0.2 ng/ml and 5.6 +/- 0.4 ng/ml in EF and LF gilts, respectively, during the fourth h after NAL. Naloxone treatment failed to alter serum LH concentrations in EF, LF, or OVX gilts and PRL concentrations in OVX gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of the orally active progestin, Regu-mate, to overcome confinement-induced delayed puberty in gilts

Thirty-three crossbred gilts that were raised in total confinement were randomly allotted to two adjacent pens in a finishing unit at 144.7 +/- .5 days of age and 58.0 +/- 1.7 kg body weight. At approximately 253 days of age, 16 gilts were group fed a daily dose of 20 mg of Regu-mate per gilt for 18 days and 17 control gilts were group fed the same diet without Regu-mate for 18 days. Ovarian morphology was examined on 11 or 12 days after the last feeding of Regu-mate. Based on estrous behavior and ovarian morphology only one Regu-mate gilt displayed an estrus but did not ovulate and only three of the control gilts displayed estrus and ovulated at least once before the start of treatment. Three of the 16 Regu-mate gilts displayed estrus and ovulated 7.3 +/- .3 days after the last feeding and within the same time period the 3 control gilts, which previously displayed estrus, continued to have estrous cycles. One additional control gilt displayed estrus and ovulated 5 days after the last feeding of the control diet. Therefore, the proportion of gilts that displayed estrus and ovulated by the end of the experimental period were similar for the treated (25.0%) and control (23.5%) groups. Based on these results we conclude that treatment of gilts in a state of confinement-induced delayed puberty with 20 mg Regu-mate daily for 18 days failed to result in a synchronized onset of puberty in a significant proportion of gilts.     

Prostaglandin E2 counteracts the effects of PGF2 alpha in indomethacin treated cycling gilts

Twenty crossbred gilts with at least 2 consecutive estrous cycles of 18 to 21 days in length were used to study the effects of prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) on luteal function in indomethacin (INDO) treated cycling gilts. Intrauterine and jugular vein catheters were surgically placed before day 7 of the treatment estrous cycle and gilts were randomly assigned to 1 of 5 treatment groups (4/group). With exception of the controls (Group I) all gilts received 3.3 mg/kg INDO every 8 h, Groups III, IV and V received 2.5 mg PGF2; 2.5 mg PGF2 alpha + 400 micrograms PGE2 every 4 hr, or 400 micrograms PGE2 every 4 h, respectively. All treatments were initiated on day 7 and continued until estrus or day 23. Jugular blood for progesterone analysis was collected twice daily from day 7 to 30. Estradiol-17 beta (E2-17 beta) concentrations were determined in samples collected twice daily, from 2 d before until 2 d following the day of estrus onset. When compared to pretreatment values, estrous cycle length was unaffected (P greater than 0.05) in Group I, prolonged (P less than 0.05) in Groups II, IV and V; and shortened (P less than 0.05) in Group III. The decline in plasma progesterone concentration that normally occurs around day 15 was unaffected (P greater than .05) in Group I; delayed (P less than 0.05) in Groups II, IV and V; and occurred early (P less than 0.05) in Group III. Mean E2-17 beta remained high (31.2 +/- 4.9 to 49.3 +/- 3.1 pg/ml) in Groups III and IV, while the mean concentrations in Groups III and V varied considerably (17.0 +/- 2.0 to 52.2 +/- 3.5 pg/ml). The results of this study have shown that PGE2 will counteract the effects of PGF2 alpha in INDO treated cycling gilts. The inclusion of PGF2 alpha appeared to either stimulate E2-17 beta secretion or maintain it at a higher level than other treatments.     

The effects of zearalenone on reproduction in swine. I. The relationship between ingested zearalenone dose and anestrus in non-pregnant, sexually mature gilts

Ninety-nine sexually mature, non-pregnant gilts were checked for estrus daily with a mature boar and then allocated at estrus (D O) to receive 2 kg/d of a diet containing 0, 1, 5 or 10 ppm purified zearalenone between D 5 and 20 of the estrous cycle during two seasons of the year (winter and summer). None of the gilts exhibited any visual signs of "hyperestrogenism" and there was no effect of season on interestrous interval (P > 0.05). A significant effect of zearalenone dose on inter-estrous interval was detected (P < 0.001). Gilts receiving 0 or 1 ppm had similar inter-estrous intervals (21.0 +/- 0.3 and 21.5 +/- 0.8 d, respectively) whereas gilts receiving 5 and 10 ppm had extended cycles (29.2 +/- 2.9 and 32.7 +/- 3.3 d, respectively). Plasma progesterone concentrations at D 19 to 21 were higher in gilts with extended cycles (P < 0.001) and corpora lutea (CL) were present at laparotomy. Some 86% of these retained CL underwent spontaneous regression resulting in the onset of estrus within the next 30 d. Fecal zearalenone concentrations rose during ingestion of contaminated diets and declined to pretreatment values within 2 d (1 ppm) to 8 d (10 ppm) of the cessation of treatment. These data show that feeding zearalenone at concentrations of 5 to 10 ppm from D 5 to 20 of the estrous cycle causes luteal maintenance and extended inter-estrous intervals. Spontaneous regression of these CL usually occurs within 30 d after zearalenone is removed from the diet. Fecal zearalenone analysis does not appear to be an effective method for determining prior exposure to zearalenone when carried out more than a few days following the last ingestion of zearalenone.     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

Maturation, fertilization and complete development of porcine oocytes matured under different systems

This study was designed 1) to determine the effectiveness of 2 in vitro maturation systems commonly employed to produce nuclear and cytoplasmically mature pig oocytes, 2) to assess the effects of boar, sperm concentration and maturation system on oocyte penetrability and male pronucleus formation and 3) to determine the ability of the in vitro matured oocytes to be fertilized in vivo by artificial insemination (AI) of sows. The differences examined between the 2 maturation systems included the culture medium (Waymouth vs TCM199), hormones, additives, culture conditions (static vs gentle agitation) presence or absence of porcine follicular fluid (PFF) and presence or absence of follicular shells. The results showed that nuclear maturation rate was similar in both systems (83.3 +/- 3.5 vs 86.4 +/- 2.5%), and intracellular content of glutathione was 5.21 +/- 0.73 vs 3.5 +/- 0.39 pmol/oocyte, although no correlation between these parameters was observed. The penetration rate and number of sperm cells per oocyte were dependent on the boar, maturation system and sperm concentration, but the rate of male pronuclear formation seemed to be influenced only by the boar and the maturation system but not by sperm concentration. In vivo fertilization of in vitro matured oocytes showed that both maturation systems could yield viable oocytes since 3 of 4 gilts and 2 of 4 gilts, respectively, became pregnant. Failure to become pregnant was not associated with inadequate oocyte maturation since control gilts, which received their own ovulated oocytes rather than in vitro matured oocytes at transfer, also did not become pregnant. We conclude that polyspermy may be an inherent problem in the IVF but not in the IVM systems.     

Effects of injection of hcg during the estrous cycle on follicle development and the inter-estrous interval

Pseudopregnancy in pigs can be induced by the administration of a single dose of hCG at Day 12 of the estrous cycle. However, the resulting length of pseudopregnancy can be extremely variable. In this study, it was investigated whether time of hCG administration (day of the cycle) and degree of follicle growth after hCG administration were related to the length of inter-estrous interval (pseudopregnancy). In the first experiment, groups of cyclic gilts were given 1500 IU hCG at either Day 11 (D 11; n=14) or Day 12 (D12; n=14) after onset of estrus, or not treated (Control; n=13). Follicle development was assessed daily using transcutaneous ultrasonography. Follicle size in the Control gilts remained relatively constant between Days 11 and 17, whereas in the treated gilts, follicle size increased (P < 0.001) within 4 days (D11) and 2 days (D12) after treatment. The inter-estrous interval was increased (P < 0.01) in the hCG-treated gilts (34.7+/-6.3 and 37.6+/-11.1 days in the D11 and D12 gilts, respectively), compared to Controls (22.3+/-5.2 d). About two-thirds of the treated gilts returned to estrus between Days 32 and 39 after onset of first estrus. No relationships were found between follicle development after treatment and length of the inter-estrous interval. In a second experiment, 16 cyclic gilts were treated with 1500 IU hCG at Day 12 and Day 15 of the estrous cycle. Follicle development was assessed at Days 12, 15 and 18. At Day 18, average follicle size was 8.4+/-2.0 mm. The inter-estrous interval was 39.7+/-5.4 days and 14 of 16 gilts returned to estrus between Days 34 and 44 after onset of first estrus. Again, no relationships were found between follicle development after treatment and the duration of the inter-estrous interval. We conclude that, based on the duration of the inter-estrous interval, administration of hCG during the luteal phase induced a short pseudopregnancy. However, the induction of accessory corpora lutea or follicular luteinization cannot be discounted.     

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.     

High overall in vitro efficiency of porcine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture

We investigated the in vitro developmental competence of porcine embryos produced from in vitro matured (IVM) oocytes by improved HMC and parthenogenetic activation (PA). Embryos were cultured in a modified North Carolina State University (NCSU37) medium. Firstly, we compared the developmental competence between oocytes from sows and gilts by zona-intact (ZI) and zona-free (ZF) PA. Significantly higher (p < 0.05) blastocyst rates were obtained from sow oocytes (42 +/- 4% for ZF and 55 +/- 6% for ZI) than gilt oocytes (20 +/- 2% for ZF and 26 +/- 5% for ZI). Secondly, sow oocytes were used to establish the modified HMC that was based on a modified enucleation with partial zona digestion and trisection of porcine oocytes and the use of three cytoplasts and one somatic cell for embryo reconstruction. In vitro fertilization (IVF) and in parallel ZF PA were used as the control systems. After oocyte trisection, >90% of oocyte fragments were recovered, resulting in an average of 37 reconstructed embryos from 100 oocytes. Blastocyst rates of HMC, IVF, and ZF PA embryos were 17 +/- 4%, 30 +/- 6%, and 47 +/- 4%, respectively. Our results prove that HMC in pigs may result in high in vitro efficiency up until the blastocyst stage. In vivo developmental competence will be confirmed in embryo transfer experiments.     

Prostaglandin-induced regression of porcine corpora lutea maintained by estrogen.

Corpora lutea were marked with suture in 24 crossbred gilts on day 7 to 9 of the estrous cycle (first day of estrus equals 0). All gilts were injected with 5 mg of estradiol benzoate (EB) daily from day 10 to 15 to extend the lifespan of corpora tutea, then the gilts were randomly assigned to two groups. On day 20, the 12 gilts of Group 1 were injected with 10 mg PGF-2ALPHA, and the 12 gilts of Group 2 were injected with saline. Ovaries were recovered 10 to 13 days after PGF-2ALPHA or saline injection. Ten gilts in Group 1 displayed estrus 5 plus or minus 0.7 days after PGF-2ALPHA injection, but only two gilts in Group 2 displayed estrus during the experimental period. In gilts that displayed estrus, all marked CL had regressed. Marked CL were still present in all 12 gilts that failed to exhibit estrus during the experimental period. These results show that in the pig, PGF-2ALPHA caused regression of CL that were maintained beyond the normal luteal phase of the estrous cycel by EB treatment.     

Endocrine parameters of human follicular fluid and fertilization capacity of oocytes

The induction of multiple follicular growth during ovarian stimulation for in vitro fertilization (IVF) implies follicular asynchrony. As a consequence oocytes of different quality are obtained. The maturity and fertilizability of oocytes cannot sufficiently be predicted by their morphological appearance under the light microscope. Looking for additional parameters of oocyte quality, FSH, hCG, estradiol (E2), progesterone (P), testosterone (T), prolactin (PRL) and cAMP were analysed in human follicular fluid (FF) containing a morphologically mature oocyte. The evaluation of the relationship between FF values and oocyte fertilization showed the following results: no differences of FSH, hCG, E2, P and T concentrations in FF between the group of fertilized and not fertilized ova. However, significant differences were detected for PRL and cAMP. In FF of fertilized oocytes PRL content was higher (38.8 +/- 2.2 vs 29.7 +/- 2.3 ng/ml, P less than 0.01) and cAMP level was lower (32.7 +/- 1.9 vs 59.8 +/- 7.4 pmol/ml, P less than 0.01) as compared with FF of unfertilizable oocytes. In conclusion PRL- and cAMP concentration of FF might be additional parameters of oocyte maturation and fertilizability.     

The effects of administration of gonadotrophins and estrogens on prenatal survival in gilts

In gilts ovulation occurs over a 4 to 8-hour period, with 70% of the ova being shed over a relatively short span of time. These oocytes supposedly give rise to more developed embryos at Days 10 to 12 which advance the uterine environment and reduce survival rates of less developed embryos because of an asynchronous environment. The aim of this experiment was to reduce embryo mortality by influencing the duration and pattern of ovulation. Crossbred gilts (n = 98) were bred at their first observed estrus after being exposed to boars at 200 days of age. Estrus detection was carried out daily at 0000, 0800 and 1600 hours. All gilts were artifically inseminated with fresh semen, with a minimum of 2.7 billion spermatozoa, at both 16 and 32 hours after detection of estrus. Gilts were randomly assigned to one of the following treatments at detection of estrus: 1) 500 IU (2ml) chorionic gonadotrophin (hCG) injected intravenously at the onset of estrus (n = 22); 2) 16 mug (4 ml) gonadotrophin releasing hormone (GnRH) injected intravenously at the onset of estrus (n = 25); 3) 11.5 mug estrogen added to the semen at the time of AI (n = 25); 4) control, untreated gilts (n = 26). All gilts were slaughtered at Day 30 of gestation (Day 0 = day of detected estrus). The mean (+/-SEM) number of ovulations in pregnant gilts per treatment was 13.0 +/- 0.52, 12.6+/-0.51, 13.6+/-0.54 and 13.3+/-0.52, while the mean (+/-SEM) number of normal embryos per treatment was 10.3+/-0.67, 10.5+/-0.66, 10.3 +/- 0.69 and 10.5 +/- 0.67 for hCG, GnRH, estrogen and control groups, respectively, for an embryonic survival rate of 80 +/- 4.2%, 83 +/- 4.1%, 74 +/- 4.3% and 79+/-4.2% in pregnant gilts. If nonpregnant gilts are included, the embryonic survival rate for treatments 1 to 4 was 76+/-7.0%, 73+/-6.5%, 60+/-6.5%, and 64+/-6.4%, respectively. There was no significant difference between treatments for any of these variables. There was no evidence that administration of hCG, or GnRH at the onset of estrus, or the addition of estrogen to semen improved embryonic survival in gilts by Day 30 in this experiment.     

Concentrations of 13, 14-dihydro-15-keto-prostaglandin F(2)alpha, estradiol-17beta and progesterone during the peripubertal period in heifers

Twenty prepubertal Holstein heifers were utilized to assess plasma 13, 14-dihydro-15-keto-prostaglandin F(2)alpha (PGFM), serum progesterone (P(4)) and estradiol-17beta (E(2)) concentrations as well as the E(2):P(4) ratio during the onset of puberty in cattle. All animals were maintained as a group along with a sterile marker bull to assist in the detection of estrus. Upon detection of the first estrus (Day=O), daily blood samples were collected from a jugular vein until the heifers had completed 3 estrous cycles. The average body weight and age at first estrus were 247.6+/-4.8 kg and 304.0+/-7.5 days, respectively. Frequency of abnormal length estrous cycles was greater (P<0.02) during the first (40%) and second (35%) cycles than during the third estrous cycle (0%). All heifers had normal cycle lengths (18 to 24 days) by the third estrous cycle. Serum P(4) was greater during the third cycle (P<0.05) from Day 10 to Day 4 before the next estrus compared with the same period of the first estrous cycle. Serum E(2) did not peak until the day of estrus in the first cycle, whereas E(2) reached a maximal level 2 days before estrus in the third estrous cycle. Serum E(2) was higher (P<0.0001) 2 days before estrus in the third cycle than in the first estrous cycle. Plasma PGFM reached maximum concentrations 3 days before estrus in the third cycle compared with 1 day before estrus at the end of first estrous cycle. As estrus approached during the third cycle, PGFM rose 1 day before E(2) rose and P(4) declined, while the rise in PGFM and E(2) occurred simultaneously, with P(4) declining at the end of the first estrous cycle. During diestrus, the E(2):P(4) ratio was lower (P<0.07) in the third cycle than in the first, but it was higher (P<0.04) at estrus and 1 day before in the third estrous cycle. These data reveal a high incidence of abnormal length estrous cycles during the first two estrous cycles of the peripubertal period, and demonstrate anomalies in uterine and ovarian endocrine activity during the peripubertal period in cattle.     

Influence of elevated ambient temperature after breeding on plasma corticoids, estradiol and progesterone in gilts

Thirty Yorkshire gilts were used to determine the influence of elevated ambient temperature during days 8 to 16 after breeding on concentrations of progesterone, corticoids and estradiol in plasma. Gilts were mated to a boar between 0800 and 1000 hr of the first day (day 0) and each subsequent day of estrus. On day 5 or 6, 22 gilts were anesthetized and a cannula was placed in the anterior vena cava. Gilts were randomly allotted to either control (23+/-1 C) or hot (35+/-1 C for 12 hr and 32+/-1 C for 12 hr daily) environmental chambers on day 8. Gilts were bled twice daily (0800 and 2000 hr) on days 9 through 16, then once daily until day 28. A third group of noncannulated gilts was bred and assigned to the control chamber. All cannulated gilts were injected intravenously with 25 IU of ACTH on day 16, and frequent blood samples were collected. Concentrations of progesterone in plasma were similar for heat-stressed and control gilts that were pregant. However, progesterone in plasma was reduced during days 13 to 19 in nonpregnant heat-stressed gilts compared to the pregnant and nonpregnant control gilts. Concentrations of estradiol in plasma were greater in nonpregnant heat-stressed gilts than in nonpregnant control and all pregnant gilts on days 10, 11 and 12 after estrus. Concentrations of corticoids and progesterone in plasma after infusion of ACTH on day 16 after breeding were reduced in heat-stressed nonpregnant gilts compared to heat-stressed pregnant, control pregnant and control nonpregnant gilts. These data indicate that reduced reproductive performance which occurs after exposure of gilts to increased ambient temperature during days 8 to 16 after breeding may be related to altered endocrine function.     

Sustained but not repeated acute elevation of cortisol impaired the luteinizing hormone surge, estrus, and ovulation in gilts.

We tested the hypothesis that sustained and repeated acute elevation of cortisol would impair the LH surge, estrus, and ovulation in gilts. Cortisol was injected intramuscularly, to achieve a sustained elevation of plasma concentrations of cortisol, or intravenously, to achieve an acute elevation of plasma concentrations of cortisol. Control gilts received i.m. injections of oil and i.v. injections of saline. These treatments were administered to gilts (n = 6 per treatment) at 12-h intervals from Days 7 to 11 of the estrous cycle until after estrus ceased or until Day 27 or 28 of the estrous cycle, whichever came first. The repeated acute elevation of cortisol had no effect on the LH surge, estrus, or ovulation. In contrast, when the elevation of cortisol was sustained, the LH surge, estrus, and ovulation were inhibited. We conclude that cortisol is capable of direct actions to impair reproductive processes in female pigs but that plasma concentrations of cortisol need to be elevated for a substantial period for this to occur.     

Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 +/- 2.1 pmol/oocyte) and medium (13.69 +/- 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 +/- 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 +/- 5.18 nM and 52.25 +/- 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 +/- 68.6 ng/ml) than from medium (40.0 +/- 6.4 ng/ml) and small (41.2 +/- 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 +/- 45.9 ng/ml) and medium (267.5 +/- 38.6 ng/ml) follicles were significantly higher than that (174.7 +/- 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.