Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock.

Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus-oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.

Effects of rumen-protected methionine and choline supplementation on the preimplantation embryo in Holstein cows.

Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate.

Developmental programming in the preimplantation period: can it be exploited to enhance postnatal function in cattle?

The concept of developmental programming states that the function of an adult animal depends on environmental conditions to which it was exposed to before birth. Developmental programming can occur in the preimplantation period. Accordingly, certain environmental signals, acting either on the mother (for pregnancies established in vivo) or on the embryo directly (for cultured embryos), can program development of the preimplantation embryo to have effects on postnatal life. It is proposed that research on developmental programming in cattle could lead not only to elimination of adverse outcomes associated with in vitro production of embryos but also to discovery of approaches to produce a neonatal animal with superior prospects for achieving optimal production later in life.

Developmental programming in the preimplantation period: can it be exploited to enhance postnatal function in cattle?

The concept of developmental programming states that the function of an adult animal depends on environmental conditions to which it was exposed to before birth. Developmental programming can occur in the preimplantation period. Accordingly, certain environmental signals, acting either on the mother (for pregnancies established in vivo) or on the embryo directly (for cultured embryos), can program development of the preimplantation embryo to have effects on postnatal life. It is proposed that research on developmental programming in cattle could lead not only to elimination of adverse outcomes associated with in vitro production of embryos but also to discovery of approaches to produce a neonatal animal with superior prospects for achieving optimal production later in life.(AU)

Cellular and molecular basis of therapies to ameliorate effects of heat stress on embryonic development in cattle

Much of the effect of heat stress on establishment and maintenance of pregnancy involves changes in ovarian function and embryonic development that reduce the competence of the oocyte to be fertilized and the resultant embryo to develop. There are three possible therapeutic approaches to manipulate the connection between hyperthermia and cellular responses to elevated temperature to improve fertility during heat stress. Embryo transfer is based on the idea that 1) most effects of heat stress on fertility involve actions during folliculogenesis or on cleavagestage embryos and 2) the embryo has acquired resistance to elevated temperature by the time it is transferred at t he morula or blastocyst stage. The mechanisms for acquis ition of thermotolerance involve changes in production of reactive oxygen species in response to heat shock as well as accumulation of antioxidants in the embryo. Synthesis of heat shock proteins may not be the controlling factor for acq uisition of thermotolerance because transcript abundance for HSPA1A and HSP90AA1 is higher for the two-cell embryo than morula. Involvement of reactive oxygen species in actions of elevated temperature on embryo survival is indicative that provision of ant ioxidants to heat-stressed cows could improve fertility. More work is needed but there are indications that pregnancy rates can be improved by feeding supplemental β-carotene or administration of melatonin implants. It is also evident that there are genes that control thermoto lerance at the cellular level. Brahman, Nelore and Romosinuano embryos have increased resistance to heat shock as compared to Holstein or Angus embryos. Mutations in the gene for heat shock protein 70 that control resistance of cells t o heat shock have been identified in Holsteins. Selection for the desirable alleles of genes conferring cellular thermotolerance could lead to development of strains of cattle whose fertility is resistant to disruption by heat stress. Pursuing these and other therapeutic approaches for reducing consequences of heat stress for livestock species should be a priority because of the prospects for continuing global climate change.(AU)

Cellular and molecular basis of therapies to ameliorate effects of heat stress on embryonic development in cattle

Much of the effect of heat stress on establishment and maintenance of pregnancy involves changes in ovarian function and embryonic development that reduce the competence of the oocyte to be fertilized and the resultant embryo to develop. There are three possible therapeutic approaches to manipulate the connection between hyperthermia and cellular responses to elevated temperature to improve fertility during heat stress. Embryo transfer is based on the idea that 1) most effects of heat stress on fertility involve actions during folliculogenesis or on cleavagestage embryos and 2) the embryo has acquired resistance to elevated temperature by the time it is transferred at t he morula or blastocyst stage. The mechanisms for acquis ition of thermotolerance involve changes in production of reactive oxygen species in response to heat shock as well as accumulation of antioxidants in the embryo. Synthesis of heat shock proteins may not be the controlling factor for acq uisition of thermotolerance because transcript abundance for HSPA1A and HSP90AA1 is higher for the two-cell embryo than morula. Involvement of reactive oxygen species in actions of elevated temperature on embryo survival is indicative that provision of ant ioxidants to heat-stressed cows could improve fertility. More work is needed but there are indications that pregnancy rates can be improved by feeding supplemental β-carotene or administration of melatonin implants. It is also evident that there are genes that control thermoto lerance at the cellular level. Brahman, Nelore and Romosinuano embryos have increased resistance to heat shock as compared to Holstein or Angus embryos. Mutations in the gene for heat shock protein 70 that control resistance of cells t o heat shock have been identified in Holsteins. Selection for the desirable alleles of genes conferring cellular thermotolerance could lead to development of strains of cattle whose fertility is resistant to disruption by heat stress. Pursuing these and other therapeutic approaches for reducing consequences of heat stress for livestock species should be a priority because of the prospects for continuing global climate change.

Challenges to fertility in dairy cattle: from ovulation to the fetal stage of pregnancy / Desafios na fertilidade de gado leiteiro: da ovulação ao estágio fetal da gestação

One critical determinant of fertility is the establishment and maintenance of pregnancy after insemination or embryo transfer. Pregnancy success is low in lactating dairy cows. Reduced fertility of the dairy female is only expressed in the lactational state but the magnitude of depression of fertility is not related to milk yield. Pregnancy failure occurs throughout gestation with the greatest frequency of pregnancy losses being in the period from fertilization to day 5-7 after insemination. Poor fertility in lactating cows represents, in part, failure of genetic selection for reproductive traits. In addition, the uterus of the lactating cow is less able to support embryonic and fetal development than the uterus of the non-lactating female. There is little evidence that the oocyte is compromised in lactating cows. Among the determinants of pregnancy success in lactating cows is progesterone secretion in the post-ovulatory period, environment of the ovulatory follicle during its terminal growth, energy balance, and amounts of specific nutrients in the diet. Progress in improving reproductive function of the lactating cow is being made and the historical decline in reproductive function is beginning to be reversed.(AU)

Um determinante crítico da fertilidade é o estabelecimento e manutenção da gestação após inseminação ou transferência de embrião. O sucesso na gestação é baixo em animais lactantes. A fertilidade reduzida da vaca leiteira é expressa apenas no estádio de lactação, mas a magnitude da diminuição da fertilidade não está relacionada à produção de leite. A falha na gestação ocorre ao longo da gestação com a maior frequência de perda de gestação no período entre a fertilização e o dia 5-7 após inseminação. Fertilidade pobre em vacas lactantes representa, em parte, a falha da seleção genética para traços de reprodução. Além disto, o útero da vaca lactante é menos capaz de suportar desenvolvimento do embrião e do feto que o útero de uma fêmea não lactante. Há pouca evidência de que o oócito seja comprometido em vacas lactantes. Entre os determinantes de sucesso na gestação de vacas lactantes está o crescimento, equilíbrio energético e quantidade de ingredientes específicos na dieta. O progresso na melhoria da função reprodutiva da vaca lactante está sendo feito, e o declínio histórico na função reprodutória está começando a ser revertido.(AU)

Challenges to fertility in dairy cattle: from ovulation to the fetal stage of pregnancy / Desafios na fertilidade de gado leiteiro: da ovulação ao estágio fetal da gestação

One critical determinant of fertility is the establishment and maintenance of pregnancy after insemination or embryo transfer. Pregnancy success is low in lactating dairy cows. Reduced fertility of the dairy female is only expressed in the lactational state but the magnitude of depression of fertility is not related to milk yield. Pregnancy failure occurs throughout gestation with the greatest frequency of pregnancy losses being in the period from fertilization to day 5-7 after insemination. Poor fertility in lactating cows represents, in part, failure of genetic selection for reproductive traits. In addition, the uterus of the lactating cow is less able to support embryonic and fetal development than the uterus of the non-lactating female. There is little evidence that the oocyte is compromised in lactating cows. Among the determinants of pregnancy success in lactating cows is progesterone secretion in the post-ovulatory period, environment of the ovulatory follicle during its terminal growth, energy balance, and amounts of specific nutrients in the diet. Progress in improving reproductive function of the lactating cow is being made and the historical decline in reproductive function is beginning to be reversed.

Um determinante crítico da fertilidade é o estabelecimento e manutenção da gestação após inseminação ou transferência de embrião. O sucesso na gestação é baixo em animais lactantes. A fertilidade reduzida da vaca leiteira é expressa apenas no estádio de lactação, mas a magnitude da diminuição da fertilidade não está relacionada à produção de leite. A falha na gestação ocorre ao longo da gestação com a maior frequência de perda de gestação no período entre a fertilização e o dia 5-7 após inseminação. Fertilidade pobre em vacas lactantes representa, em parte, a falha da seleção genética para traços de reprodução. Além disto, o útero da vaca lactante é menos capaz de suportar desenvolvimento do embrião e do feto que o útero de uma fêmea não lactante. Há pouca evidência de que o oócito seja comprometido em vacas lactantes. Entre os determinantes de sucesso na gestação de vacas lactantes está o crescimento, equilíbrio energético e quantidade de ingredientes específicos na dieta. O progresso na melhoria da função reprodutiva da vaca lactante está sendo feito, e o declínio histórico na função reprodutória está começando a ser revertido.

Pregnancy success of lactating Holstein cows after a single administration of a sustained-release formulation of recombinant bovine somatotropin.

BACKGROUND: Results regarding the use of bovine somatotropin for enhancing fertility in dairy cattle are variable. Here, the hypothesis was tested that a single injection of a sustained-release preparation of bovine somatotropin (bST) during the preovulatory period would improve pregnancy success of lactating dairy cows at first service. RESULTS: The first experiment was conducted in a temperate region of Mexico. Cows inseminated following natural estrus or timed artificial insemination were given a single injection of bST or a placebo injection at insemination (n = 100 cows per group). There was no significant difference between bST and control groups in the proportion of inseminated cows diagnosed pregnant (29 vs 31% pregnant). The second experiment was performed during heat stress in Florida. Cows were subjected to an ovulation synchronization regimen for first insemination. Cows treated with bST received a single injection at 3 days before insemination. Controls received no additional treatment. As expected, bST did not increase vaginal temperature. Treatment with bST did not significantly increase the proportion of inseminated cows diagnosed pregnant although it was numerically greater for the bST group (24.2% vs 17.8%, 124-132 cows per group). There was a tendency (p = 0.10) for a smaller percent of control cows to have high plasma progesterone concentrations (>/= 1 ng/ml) at Day 7 after insemination than for bST-treated cows (72.6 vs 81.1%). When only cows that were successfully synchronized were considered, the magnitude of the absolute difference in the percentage of inseminated cows that were diagnosed pregnant between bST and control cows was reduced (24.8 vs 22.4% pregnant for bST and control). CONCLUSION: Results failed to indicate a beneficial effect of bST treatment on fertility of lactating dairy cows.

The effect of bovine interferon-alpha I1 on pregnancy rate in heifers.

Bovine interferon-alpha I1 (bIFN-alpha) may be useful for enhancing fertility in sheep and cattle because it has extensive sequence homology with ovine and bovine trophoblast protein-1 and, like those proteins, extends corpus luteum lifespan. To test the effectiveness of bIFN-alpha to enhance fertility, several experiments were performed in which inseminated heifers were given i.m. injections of bIFN-alpha approximately at the time of embryo-mediated signals that result in maintenance of the corpus luteum. In Exp. 1, heifers given 20 mg of bIFN-alpha daily from d 14 to 17 tended (P less than .07) to have lower pregnancy rates at d 110 to 112 of gestation (36/75; 48% vs 43/72; 60%). Similar results were obtained in Exp. 2 when heifers received a single injection of 40 mg of bIFN-alpha or placebo at d 13 after estrus; pregnancy rates at d 42 were 39/104 (38%) for bIFN-alpha and 47/98 (48%) for placebo. In Exp. 3, heifers were given gradually increasing doses of bIFN-alpha or placebo from d 11 to 19, because such a regimen had been shown to reduce the number of heifers experiencing hyperthermia after bIFN-alpha injection. Pregnancy rates were 42/95 (44%) for bIFN-alpha and 62/111 (56%) for placebo. Across all three experiments, pregnancy rates were lower (P less than .01) for heifers treated with bIFN-alpha (117/274; 43%) than for heifers treated with placebo (152/281; 54%). In conclusion, these results demonstrate that, under the administration systems used, bIFN-alpha does not increase pregnancy rate, but rather tends to reduce it.

Possible mechanisms for reduction of circulating concentrations of progesterone by interferon-alpha in cows: effects on hyperthermia, luteal cells, metabolism of progesterone and secretion of LH.

Experiments were performed to determine the mechanism by which recombinant bovine interferon-alpha I1 (rbIFN-alpha) causes an acute reduction in plasma concentrations of progesterone. In experiment 1, administration of a prostaglandin synthesis inhibitor blocked rbIFN-alpha-induced hyperthermia but did not prevent the decline in plasma concentrations of progesterone. The decline in progesterone concentrations caused by rbIFN-alpha was, therefore, not a direct consequence of the associated hyperthermia or of pathways mediated through prostaglandin synthesis. It is also unlikely that rbIFN-alpha acts to increase the clearance of progesterone since injection of rbIFN-alpha did not decrease plasma concentrations of progesterone in ovariectomized cows given an intravaginal implant of progesterone (experiment 2). In experiment 3, rbIFN-alpha did not affect basal and LH-induced release of progesterone from cultured luteal slices, indicating that rbIFN-alpha is unlikely to affect luteal function directly. Injection of rbIFN-alpha did, however, cause a decrease in plasma concentrations of LH in ovariectomized cows (experiment 4) that coincided temporally with the decrease in progesterone concentrations seen in cows having a functional corpus luteum. The present results strongly suggest that rbIFN-alpha acts to reduce secretion of progesterone by interfering with pituitary support for luteal synthesis of progesterone. The finding that rbIFN-alpha can inhibit LH secretion implies that interferon-alpha molecules should be considered among the cytokines that can regulate hypothalamic or pituitary function.

Regulation of bovine endometrial secretion of prostaglandins and synthesis of 2',5'-oligoadenylate synthetase by interferon-alpha molecules.

Experiments were performed to (1) verify the inhibitory effect of bovine trophoblast protein-1 (bTP-1) on uterine prostaglandin synthesis, (2) evaluate whether other interferon-alpha (IFN-alpha) molecules also inhibit prostaglandin secretion, and (3) test whether the enzyme 2',5'-oligoadenylate synthetase (2-5A synthetase) can be induced in endometrium by interferon-alpha. In experiment 1, all interferon molecules (bTP-1, oTP-1, bIFN-alpha and hIFN-alpha) equally inhibited secretion of PGF and PGE2 from endometrial explant cultures obtained at day 17 of the estrous cycle. In experiment 2, endometrial explants obtained from day 17 of the cycle were cultured with and without bovine serum albumin (BSA; 50 micrograms/ml) and bIFN-alpha (0, 0.84, 4.2, and 42 nM). Addition of BSA to the culture medium greatly enhanced the accumulation of PGF into the medium. The bIFN-alpha inhibited accumulation of PGF and PGE2 in both the presence or absence of BSA by 12 h. All three concentrations of bIFN-alpha were equally effective in inhibiting prostaglandin accumulation. Additionally, all concentrations of bIFN-alpha increased the amounts of 2-5A synthetase in endometrium. In conclusion, these results confirm the inhibitory effect of bTP-1 on PGF release from endometrium and demonstrate that bTP-1 can also inhibit PGE2 secretion. Furthermore, other interferon-alpha molecules, including bIFN-alpha, hIFN-alpha, and oTP-1, also reduced PGF and PGE2 secretion in culture. It is likely, therefore, that conceptus and other interferon-alpha molecules exert similar effects on endometrium in vitro and that the antiluteolytic effects of bIFN-alpha in vivo are mediated in part by changes in endometrial prostaglandin synthesis.

Rescue of the Corpus luteum from luteolysis by bovine trophoblast protein-1: an example of maternal recognition of pregnancy

Bovine trophoblast protein-1 (bTP-1) is an interferon molecule produced by trophectoderm tissue that plays a critical role in maintenance of luteal function during early pregnancy. Synthesis and secretion of bTP-1 begins as early as day 10 of pregnancy, is maximal around days 15-22, and continues to at least day 25. Bovine trophoblast protein-1 exists as a complex of up to 13 microheterogeneous glycoproteins in three molecular weight classes (21-22 kDa, 23-24 kDa and 26 kDa), with each molecular weight class having 4-5 isoelectric variants of pI 5.6-6.6. The several isoelectric variants of bTP-1 arise from different mRNA and the different size classes from differences in degree of glycosylation. As for other interferon-* molecules, bTP-1 possesses antiviral activity and inhibits lymphocyte proliferation. The major role of bTP-1, however, is to block endometrial secretion of prostaglandin F2* so as to prevent luteolysis. This effect of bTP-1 may be exerted through induction of an inhibitor in endometrial epithelial cells that blocks the conversion of arachidonic acid to prostaglandins. Interestingly, other interferon-* molecules can also block secretion of endometrial PGF2* in vitro and one of these, bovine interferon-*1 1, has been shown to be effective in extending luteal lifespan when administered intramuscularly. This latter finding raises the possibility of using systemically-administered bTP-1 or other interferon-* molecules for enhancing fertility. Before such schemes can be feasible, however procedures must be found to minimize side effects of interferon administration such as hyperthermia and a transient depression in circulating progesterone concentrations. Future research on bTP-1 will concentrate on developing practical systems for fertility enhancement using bTP-1 or related interferons as well as on problems of mechanism of action of bTP-1 and regulation of expression of the bTP-1 gene in trophectoderm