BAX regulates follicular endowment in mice

It is believed that the endowment of primordial follicles in mammalian ovaries is finite. Once follicles are depleted, infertility ensues. Thus, the size of the initial endowment has consequences for fertility and reproductive longevity. Follicular endowment is comprised of various processes that culminate with the incorporation of meiosis-arrested oocytes into primordial follicles. Apoptosis is prominent during follicular endowment, and apoptosis regulatory genes are involved in its regulation. Conflicting data exist with regard to the role of the proapoptotic Bcl-2 associated X protein (BAX) in follicular endowment. Therefore, we investigated the role of BAX during follicular endowment in embryonic and neonatal ovaries. We found that BAX is involved in regulating follicular endowment in mice. Deletion of Bax yields increased oocyte numbers in embryonic ovaries and increased follicle numbers in neonatal ovaries when compared with wild-type ovaries. Increased follicular endowment in Bax -/- ovaries is not due to enhanced germ cell viability. Further, it is not due to an increased primordial germ cell (PGC) allotment, a delay in the onset of meiosis, or altered proliferative activity of oogonia. Instead, our data suggest that the regulatory activity of BAX in follicular endowment likely occurs during PGC migration, prior to PGC colonization of the gonad.     

Changes in insulin-like growth factor binding protein (IGFBP) isoforms during bovine follicular development

UThe insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and so regulate their access to the type 1 and 2 IGF receptors. This is the principal mechanism involved in regulating IGF bioavailability during folliculogenesis. IGFBPs undergo a number of post-translational modifications, including proteolytic cleavage, phosphorylation and glycosylation, which can regulate the affinity of IGFBPs for IGFs. However, the post-translational changes to IGFBPs that occur during folliculogenesis have not been fully characterized. The charge and size variants of the IGFBPs in bovine follicular fluid were examined by two-dimensional non-reducing SDS-PAGE followed by non-isotopic western ligand blot analysis, and immunoblot analysis during follicular development. The results demonstrate the presence of at least 51 IGFBP isoforms corresponding to IGFBP-1 to -6 in bovine follicular fluid from subordinate follicles, many of which were phosphorylated. The total number of IGFBPs was reduced in dominant follicles, whereas no gross changes in isoforms were observed during follicular development. These results demonstrate the high degree of conservation of IGFBP post-translational modifications between species, and from the in vitro dephosphorylation of these proteins it is hypothesized that these modifications may result in changes to IGF binding or susceptibility to proteolytic cleavage.     

Tall fescue endophyte: effect on congenital development and pup growth in mice

This study was designed to assess effects of a diet containing endophyte (Acremonium coenophialum)-infected tall fescue seed on litter growth and development in mice. Twenty pairs of mature CD-1 mice were allocated to dietary treatments containing 50% (wt/wt) rodent chow plus either 50% endophyte-infected tall fescue seed or seed that was not infected. At parturition, six pups of approximately equal BW and sex ratio were selected from each litter and fostered to each of the dams for suckling, resulting in four suckling groups. Pups born to dams fed the infected diet weighed less at birth and experienced developmental delays in BW gain and in eye opening (16.7 vs. 13.7 d) regardless of the foster dam's diet. Pups born of dams fed the diet that was not infected attained the greatest growth rates and weaning weights; those suckling dams fed the diet that was not infected demonstrated the largest gains. Pups born of and suckled by dams fed the infected diet demonstrated greatly reduced growth rates and weaning weights compared with the other groups. Results suggest that the congenital effect on pups born of dams fed an infected diet was significant in lowering the growth rate of the pups during suckling. The dam's ability to provide nourishment to the pups also was lowered when fed this diet. When these two factors were combined, the differences were greater than for either factor separately.     

Regulation of ovarian follicular growth by somatotropin and insulin-like growth factors in cattle

Somatotropin (ST), insulin-like growth factor (IGF)-I, and IGF-II affect animal growth and lactation as well as animal reproduction. Understanding the effects of ST and the IGF on reproduction is important because ST and IGF-I undergo dynamic changes prior to the postpartum breeding period. In addition, administration of recombinant bovine somatotropin (rbST) to lactating cows is a common practice that increases blood concentrations of ST and IGF-I during the breeding period. In vivo, administration of rbST caused greater ovarian follicular development. The effects of rbST may represent direct actions of ST because ST receptors are found within granulosa cells as well as oocytes. Alternatively, the actions of ST may be indirectly mediated by increased IGF-I and (or) nutrient partitioning that occurs after rbST. Both IGF-I and IGF-II are synthesized within the ovary. Ovarian IGF are, therefore, a composite of IGF from both endocrine (liver) and autocrine and paracrine (ovary) sources. The IGF stimulate ovarian function by acting synergistically with gonadotropins to promote growth and steroidogenesis of ovarian cells. Actions of IGF-I and -II are restrained by a series of IGF binding proteins (IGFBP) that either originate from the blood or are synthesized locally within the follicle. Degradation and differential synthesis of IGFBP are important mechanisms regulating IGFBP amounts. The relative amounts of IGFBP may ultimately determine ovarian IGF action. Future studies of ST and IGFs should focus on the hormones, receptors, and binding proteins as well as the metabolic requirements for normal ovarian function in dairy cattle.     

Effect of bovine follicular fluid and follicle-stimulating hormone on follicular growth in unilaterally ovariectomized prepuberal heifers

A study was conducted to determine if charcoal-extracted follicular fluid inhibits FSH-induced follicular development in prepuberal heifers. Thirty-six prepuberal heifers were allotted by breed and weight to a 2 x 2 factorial experiment involving charcoal-extracted follicular fluid and FSH treatments. Heifers were unilaterally ovariectomized and injected (intravenously; 10 ml) every 8 h for 88 h with either charcoal-extracted follicular fluid or saline. Follicle-stimulating hormone (2 mg) or saline was injected (intramuscularly) every 8-h starting 24 h after initiation of charcoal-extracted follicular fluid to 88 h following unilateral ovariectomy. Plasma samples were collected at 8-h intervals from 48 h prior to unilateral ovariectomy to 96 h following unilateral ovariectomy when the remaining ovary was removed. Follicular fluid and total ovarian weight increased following FSH treatment. The increases were not inhibited by charcoal-extracted follicular fluid. Total number of surface follicles was similar among treatments. However, FSH induced a shift in follicular diameter from small (less than or equal to 3 mm) to medium (7 to 9 mm) or large (10 to 13 mm) follicles, which was unaffected by charcoal-extracted follicular fluid. Plasma concentration of FSH, but not LH, declined following charcoal-extracted follicular fluid administration. In summary, charcoal-extracted follicular fluid did not inhibit FSH-induced follicular development in prepuberal heifers when charcoal-extracted follicular fluid was administered at a dosage that reduced circulating concentration of FSH by approximately 40%.     

Effect of direct ovarian injection of vascular endothelial growth factor gene fragments on follicular development in immature female rats

Vascular endothelial growth factor (VEGF) expression in granulosa cells is associated with the thecal vasculature growth during ovarian follicular development. We hypothesized that injection of VEGF gene fragments directly into the rat ovary would induce production of a large number of ovulatory follicles and that these follicles would ovulate. To test this hypothesis, we treated immature female rats with combinations of hormones and VEGF gene fragments. The animals were divided into two groups: one group received solution containing transfection reagents as a control (n = 5), while the other group received direct ovarian injection of VEGF gene fragments at 19 (n = 5), 21 (n = 5), 23 (n = 5), or 25 (n = 5) days after birth followed by i.p. administration of 20 IU equine chorionic gonadotropin (eCG) at the age of 26 days. Forty-eight hours after eCG injection, animals were given 20 IU human chorionic gonadotropin (hCG) i.p. and then the oocytes in both groups were counted. The maximum number of ovulated oocytes was obtained when the VEGF gene fragments were injected into the rat ovary at 21 days after birth. Histological examination revealed that the injection of VEGF gene fragments markedly increased the vascular density around the preovulatory follicles and also the number of these follicles. Our data provide the first reported evidence that most ovulatory follicles generated by injection of VEGF gene fragments are able to ovulate upon hCG treatment. These results demonstrate that injection of VEGF gene fragments directly into the ovary stimulates the development of antral follicles by inducing the formation of thecal vasculature in immature female rats.     

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.     

Genetic merit for fertility traits in Holstein cows: VI. Oocyte developmental competence and embryo development

The hypothesis of this study was that cows with good genetic merit for fertility traits (Fert+) would produce oocytes and embryos of greater quality than cows with poor genetic merit for fertility traits (Fert?) and that mRNA expression of candidate genes would reflect the observed differences in quality. The aim of the study, therefore, was to determine the effect of genetic merit for fertility traits on morphological classification and mRNA abundance of key genes in immature oocytes and cumulus cells following ovum pick-up and in embryos following superovulation, artificial insemination (AI), and uterine flushing. In experiment 1, 17 Fert+ and 11 Fert? cows, ranging from 54 to 84 d in milk, were submitted to ovum pick-up on 4 occasions during a 2-wk period. Recovered cumulus–oocyte complexes (COC) were morphologically graded. Oocytes and cumulus cells were separated, and mRNA abundance of genes associated with oocyte developmental competence was measured. There was no effect of genotype on the distribution of COC grades or on the mRNA abundance of the candidate genes in grade 1 COC. In experiment 2, 20 Fert+ and 19 Fert? cows, ranging from 71 to 189 d in milk, were submitted to superovulation and AI. The uteri of cows that responded to the superovulation protocol (17 Fert+ and 16 Fert? cows) were nonsurgically flushed 7 d postovulation. Recovered embryos were morphologically graded, and mRNA abundance of genes associated with embryo development was measured in grade 1 blastocysts. The response to the superovulation protocol was assessed by counting the number of codominant follicles on the day of AI, which was similar for both genotypes (22.0 ± 9.7 and 19.8 ± 8.2 for Fert+ and Fert? cows, respectively). There was no effect of genotype on the proportion of transferable embryos recovered or on the mRNA abundance of the candidate genes tested in the grade 1 blastocysts. Of the total embryos classified as blastocysts, however, the Fert+ cows tended to have a greater proportion of grade 1 blastocysts compared with Fert? cows (90% vs. 64%, respectively). In conclusion, genetic merit for fertility traits had a no effect on mRNA abundance of the candidate genes that were examined in immature oocytes and cumulus cells and in embryos recovered after superovulation. The observed differences in morphological blastocyst quality following superovulation would suggest that the superior reproductive performance of Fert+ cows could arise during the later stages of embryo development from d 7 until maternal recognition of pregnancy.     

Effects of IGF-I bioavailability on bovine preantral follicular development in vitro

The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P < 0.05) and increased estradiol production over control levels (P < 0.05). However, follicles treated with LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro.     

In vitro follicular development of cryopreserved mouse ovarian tissue

In a previous report, we showed that follicles isolated from frozen/thawed mouse ovarian tissues reached the mature follicle stage on the 12th day of culture. However, the developmental ability was lower than that of fresh ovarian tissue. The purpose of this study was to define a culture system with some technical modification for preantral follicles isolated from frozen/thawed ovarian tissue and to confirm cell injury. Ovaries obtained from three-week-old female mice were cryopreserved by the rapid freezing method. Preantral follicles isolated from frozen/thawed ovarian tissues were cultured for 12-16 days. The follicles were then stimulated with human chorionic gonadotropin. In vitro fertilization was performed on the released cumulus-oocyte complexes (COCs). Preantral follicle viability was assessed by supravital staining using Hoechst 33258. Using this stain cell death was found in part of the granulosa cells of a follicle obtained from frozen/thawed ovarian tissue. On the 14th and 16th days of culture, the diameters of follicles isolated from frozen/thawed ovaries were larger than on the 12th day of culture. The released COCs were fertilized and developed to the blastocyst stage in 15.8% (12/76) of the oocytes taken from the fresh group, and in 0% (0/73), 2.9% (2/69) and 19.1% (22/115) of the oocytes taken from the frozen/thawed group that had been cultured for 12, 14 and 16 days respectively. The preantral follicles isolated from frozen/thawed mouse ovarian tissues developed slowly compared with the freshly prepared preantral follicles. During prolonged culture from 12 to 16 days, these follicles obtained the potential to fertilize and develop to the blastocyst stage.     

Activin B is produced early in antral follicular development and suppresses thecal androgen production

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1-3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3-4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1-3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).     

Immunization with ovarian autoantigens leads to reduced fertility in mice following follicular dysfunction

Immunoproteomics using sera of women with ovarian autoimmune diseases such as primary ovarian insufficiency and IVF embryo transfer recruits led to identification of three proteins namely alpha actinin 4 (α-ACTN4), heat-shock 70 protein 5 (HSPA5), and actin beta (ACTB). This study deals with the establishment of a peptide ELISA for screening sera of antiovarian antibody (AOA)-positive patients and further delves into understanding the role of these three proteins in ovarian autoimmunity in a mouse model. Using in silico approach, antigenic peptides of these proteins were identified and used for peptide ELISA. ELISA results indicated that AOA-positive sera showed reactivity with only specific peptides. The functional significance of the dominant peptides was studied by active immunization of female mice with these peptides. All immunized mice generated high antibody titers and profound effect on ovaries with few primordial (2.4±0.1, 2.4±0.2, and 2±0.1), primary (2.4±0.5, 1.7±0.3, and 2.4±0.3), preantral (2.3±0.5, 3.4±0.3, and 2.9±0.3), antral (0.9±0.2, 1.6±0.8, and 2.3±0.6) follicles, and corpora lutea (2.8±0.8, 2.9±1.7, and 4.6±2.3), and increased number of atretic follicles (5.5±0.4, 4.9±1.8, and 7.5±1.0) in ACTN4-, HSPA5-, and ACTB-immunized mice compared with control animals (3.0±0.2, 3.5±0.6, 3±0.1, 3.6±0.2, 4.7±0.3, and 1.5±0.3) respectively. These mice when mated with fertile male mice showed an overall 25-43% reduction in fertility compared with controls. The data clearly suggest that the dominant antigenic epitopes of the three proteins play critical role in fertility and could possibly be the key autoimmune targets. These epitopes could be used to develop a more specific and sensitive diagnostic test for women with ovarian autoimmune diseases and to design therapy for disease management for reinstatement of ovarian function.     

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.     

Role of angiotensin in ovarian follicular development and ovulation in mammals: a review of recent advances

Angiotensin (Ang) II is widely known for its role in the control of systemic blood vessels. Moreover, Ang II acts on the vascular control of ovarian function, corpus luteum formation, and luteolysis. Over the past 10 years, our research group has been studying the new concept of the renin-angiotensin system (RAS) as an autocrine/paracrine factor regulating steroidogenesis and promoting different cellular responses in the ovary, beyond vascular function. We have developed and used different in vivo and in vitro experimental models to study the role of RAS in the ovary and a brief overview of our findings is presented here. It is widely accepted that there are marked species differences in RAS function in follicle development. Examples of species-specific functions of the RAS in the ovary include the involvement of Ang II in the regulation of follicle atresia in rats vs the requirement of this peptide for the dominant follicle development and ovulation in rabbits and cattle. More recently, Ang-(1-7), its receptor, and enzymes for its synthesis (ACE2, NEP, and PEP) were identified in bovine follicles, implying that Ang-(1-7) has an ovarian function. Other novel RAS components (e.g. (pro)renin receptor and renin-binding protein) recently identified in the bovine ovary show that ovarian RAS is poorly understood and more complex than previously thought. In the present review, we have highlighted the progress toward understanding the paracrine and autocrine control of ovarian antral follicle development and ovulation by ovarian tissue RAS, focusing on in vivo studies using cattle as a model.     

Analysis of the variations of follicular fluid composition during follicular growth and maturation in the mare using proton nuclear magnetic resonance (1H NMR)

Follicular development and ovulatory processes in mammals involve local biochemical changes as a result of substantial modifications in cellular metabolism, the most well known of which is steroid variation. In the present study, the intrafollicular variation of several other components was studied using proton nuclear magnetic resonance ((1)H NMR). This approach made it possible to demonstrate that the intrafollicular biochemical content changes during follicular growth and maturation. Follicular fluid was aspirated by ovarian puncture of the dominant follicle at various physiological stages of its development: early dominant, late dominant and preovulatory. Serum samples were collected during each puncture session. (1)H NMR was used to evaluate intrafollicular and circulating glycoconjugates (sugar chains and N-acetyl groups), lipoproteins (CH(3) and CH(2) groups), glucose metabolites (trimethylamines, acetate and lactate), amino acids (glutamine/glutamate and alanine), creatine/creatinine and polyamines. Follicular fluids were assayed by radioimmunoassay for oestradiol and progesterone contents. The intrafollicular contents of alanine and lipoproteins (CH(3) groups) decreased in the dominant follicle during growth, whereas concentrations of progesterone and oestradiol increased significantly. After injection of gonadotrophin to induce ovulation, follicular maturation was characterized by a decrease in glycoconjugates (sugar chains), trimethylamines and acetate, a decrease in oestradiol concentration, and a further increase in CH(3) groups of lipoproteins and progesterone. The results from the present study showed a clear correlation between the intrafollicular content of alanine and that of oestradiol. A correlation between progesterone and glycoconjugates (sugar chains) was also observed. Therefore, (1)H NMR was shown to be effective for studying specific changes in the biochemical composition of the follicular fluid that occur during follicular development. For the first time, the variation of several compounds (glycoconjugates, lipoproteins, glucose metabolites, amino acids and polyamines) in relation to growth and maturation was demonstrated. Some of these changes could be of crucial importance for follicular maturation and ovulation as well as for oocyte maturation and further fertilization.     

Effect of synchronization protocols on follicular development and estradiol and progesterone concentrations of dairy heifers

The objectives were to evaluate the effect of synchronization protocols on follicular development and estradiol 17-β (E₂) and progesterone (P₄) concentrations in dairy heifers. In experiment 1, 36 heifers were assigned to 1 of 6 synchronization protocols in a 3 × 2 factorial design: presynchronization with GnRH on study d −6 or −9 [study d 0 = initiation of the Cosynch + CIDR (controlled internal drug releasing insert containing P₄) protocol] or no presynchronization (control) and one injection of PGF_2α or not on study d 0. In experiment 2, 126 heifers were assigned to 1 of 4 synchronization protocols in a 2 × 2 factorial arrangement: presynchronization or not with GnRH on study d −6 and injection of PGF_2α or not on study d 0. In experiments 1 and 2, all heifers received a modified Cosynch protocol with CIDR for 7 d starting on study d 0. After the PGF_2α of the Cosynch and removal of the CIDR, heifers were detected in estrus and inseminated. Those not inseminated by study d 10 received an injection of GnRH and were timed-inseminated. Ovaries were scanned by ultrasound on d 0, 2, and 5, daily from d 7 to 14, and on d 16. Blood samples collected on d 0, 2, 7, 9, and 16 were analyzed for P₄, and the blood sample collected on d 9 was analyzed for E₂. Pregnancy was diagnosed at 28 and 40 ± 3 d after artificial insemination. In experiment 1, there was a tendency for the presynchronization protocol to affect the proportion of heifers ovulating in response to the first GnRH injection of the Cosynch + CIDR protocol. In experiment 2, a greater proportion of presynchronized heifers ovulated in response to the first GnRH injection. Although heifers receiving PGF_2α had larger ovulatory follicles on d 7 and before ovulation and shorter intervals to estrus and ovulation, these heifers tended to have decreased concentrations of E₂ during proestrus. Presynchronization of dairy heifers with GnRH increased ovulation in response to the first GnRH injection, and treatment of heifers with PGF_2α at initiation of the Cosynch + CIDR protocol increased the size of the ovulatory follicle and reduced the intervals to estrus and ovulation.     

Effects of dietary unsaturated fatty acids on oocyte quality and follicular development in lactating dairy cows in summer

Dietary sources of fatty acids were evaluated for their influence on oocyte quality and follicular development using 54 lactating cows in summer. Fat supplements were 1) sunflower oil (80% cis 18:1), 2) Ca salt of transoctadecenoic acids (57% trans 18:1), 3) Ca salt of vegetable oils (30% 18:2), and 4) linseed oil (56% 18:3 and 16% 18:2). Fats were fed at 1.35% of dietary dry matter beginning at 5 wk prior to expected calving date and at 1.5% (oils) and 1.75% (Ca salts) of dietary dry matter for 15 wk after parturition. Four days following a programmed induced ovulation, 5 transvaginal oocyte aspirations were performed 3 or 4 d apart. Three days after the last aspiration, PGF_2α was injected, followed 3 d later by a GnRH injection and a timed artificial insemination (d 0) 16 to 20 h later. For the first 4 aspirations, oocytes grading 1 or 2 were used for in vitro embryo production. Total cell number and the proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive blastomeres were analyzed at d 8. At the fifth aspiration, the occurrence of metaphase II, group II caspase activity, and TUNEL labeling were determined after oocyte maturation. A total of 1,011 oocytes were collected. The proportion of oocytes with high caspase activity was greater for grade 3 compared with grades 1 and 2 (37.5 vs. 1.54 and 1.61%). Feeding polyunsaturated fatty acids, as compared with monosaturated fatty acids, failed to affect oocyte quality, as demonstrated by subsequent embryo development. Cows fed 18:2- or 18:3-enriched diets had a larger preovulatory follicle at insemination and subsequent volume of the corpus luteum compared with those fed cis 18:1 or trans 18:1 diets (16.8, 16.2 vs. 15.0, 14.9 ± 0.7 mm; 7,323, 8,208 vs. 6,033, 5,495 ± 644 mm³, respectively). The previously documented benefits of polyunsaturated fatty acids on reproductive performance appear to reflect actions at alternative biological windows in lactating dairy cows.