Thermoregulatory response of black or red lactating Holstein cows in the hot and cold season in southern Brazil.

Dairy cows in pasture-based systems are more susceptible to heat stress. Holstein cows have the black or red phenotypes, the latter having lower absorbance of solar radiation. Therefore, the study's objective was to evaluate whether cows with red (R) coats are more resistant than black (B) cows to hot weather in a subtropical climate. R and B lactating Holstein cows were evaluated during the cold and hot seasons for internal and surface temperature and sweating rate. In the cold season, body temperature (n = 9/group) did not differ between groups, but the average superficial temperature (n = 13/group) was lower in R cows (B: 30.9 ± 0.3 °C; RW: 29.6 ± 0.3 °C; p = 0.02). In the hot season, under mild to moderate heat stress, mean body temperature (n = 9/group) of R cows was lower (B: 38.75 ± 0.01 °C; R: 38.62 ± 0.1 °C; p=<0.0001), whereas no difference was observed in superficial temperature (n = 17/group). The maximum internal temperature and sweating rate (n = 11/group), measured in the hot season, and the number of evaluations in hyperthermia in both seasons did not differ. Therefore, there were differences in thermoregulation between phenotypes under mild to moderate heat stress conditions. However, considering that only discrete differences were observed, the red and white coat is unlikely to benefit the Holstein cow's welfare under mild to moderate thermal stress.

Does GnRH treatment at TAI regardless of estrus occurrence increase pregnancy rate in crossbred Bos taurus suckled cows?

The impact of GnRH treatment on the day of TAI in beef cows has received limited investigation, especially concerning its association with estrus expression. Consequently, two experiments were conducted to assess the potential of GnRH treatment on the day of TAI to enhance fertility according to the expression or not of estrus in beef cows. Experiment 1 aimed to determine ovulation rate and luteal function, while Experiment 2 aimed to determine the effect of the two GnRH treatment approaches on pregnancy rate. In Experiment 1, multiparous Brangus suckling cows (n = 17) were submitted to an 8-day TAI protocol. Estrus occurrence was evaluated based on chalk removal on D10 (TAI) and cows were assigned to receive GnRH (25µg lecirelin; im) according to the group: GnRH (n = 7), regardless of estrus expression; or selectGnRH (n = 10), only cows not detected in estrus. Ovulation rate occurring until 77h after IVD removal did not differ (p = 0.17) between GnRH (85.7%; 6/7) and selectGnRH (100%; 10/10). Also, corpus luteum size and serum progesterone concentration were not affected (p>0.05) by treatments. In Experiment 2, crossbred taurine suckled cows (n = 384) were submitted to the same protocol as described in Experiment 1 and were randomly allocated to GnRH or selectGnRH groups. There was no difference in P/AI between groups (selectGnRH = 55.6%; GnRH = 54.3%; p = 0.7) 30 days after TAI. As expected, there was a pronounced effect (p<0.0001) of estrus expression on P/AI (Estrus = 61.5%; No estrus = 33.0%), regardless of group. In summary, ovulation timing and rate and luteal function did not differ between groups. Also, GnRH administration only in cows that do not show estrus is recommended, considering hormone savings and similar conception rate.

GnRH analogs induce a LH peak and increase pregnancy per timed-AI in ewes.

To date, there have been no studies testing the capacity of GnRH analogs and respective doses to induce a LH peak in sheep. In this sense, the present study aimed to evaluate the capacity of different synthetic forms and doses of GnRH in inducing LH release in sheep, and the effect of GnRH administration at timed artificial insemination (TAI) on pregnancy per timed-AI. In experiment 1, ewes (n = 40) received an intravaginal device (IVD) of medroxyprogesterone acetate (MPA; 60 mg) for 7 d and prostaglandin F2α analog on Day 5. On Day 7, the ewes were allocated randomly into one of eight groups (n = 5/group), which received a GnRH analog at a specific dose, as follows: lecirelin (12.5 or 25 µg), gonadorelin (50 or 100 µg), buserelin acetate (4.2 or 8.4 µg), or deslorelin (375 or 750 µg). Blood samples for LH determination were obtained at 0, 2, 4, and 6 h after GnRH and the IVDs were removed after the last blood collection. The maximal LH concentration induced by gonadorelin at doses of 50 µg and 100 µg (12.0 ± 2.4 ng/mL and 28.6 ± 7.1 ng/mL, respectively) was lower (P < 0.05) than serum LH induced by 8.4 µg of buserelin (78.9 ± 12.9 ng/mL), 375 µg and 750 µg of deslorelin (75.6 ± 7.4 ng/mL and 72.1 ± 10.6 ng/mL, respectively) and 12.5 µg and 25 µg of lecirelin (73.3 ± 17.8 ng/mL and 61.6 ± 5.9 ng/mL, respectively). However, the maximal LH concentration induced by 4.2 µg of buserelin (49.4 ± 5.9 ng/mL) was similar (P > 0.05) to the 100 µg of gonadorelin. The total release of LH (area under the curve - AUC) after treatment with 50 µg of gonadorelin (31.7 ± 5.9 ng h/mL) was lower (P < 0.05) than after other agonists. In a second experiment, 330 ewes were treated with IVD containing MPA for 7 d. Simultaneously with IVD removal, 250 µg of cloprostenol and 200 IU of eCG were administered. Then, ewes were assigned randomly to either no further treatment (control); or to receive 4.2 µg of buserelin acetate (GnRH group) at cervical TAI, which was performed with fresh semen 54 h after IVD withdrawal in all the animals. Higher pregnancy per timed-AI was observed for GnRH (50.3 %) compared to control (40.7 %). We conclude that buserelin acetate (8.4 µg), lecirelin (12.5 and 25 µg) and deslorelin (375 and 750 µg) induced a greater stimulatory effect on LH secretion than gonadorelin treatment. Furthermore, buserelin acetate treatment at TAI increased pregnancy per timed-AI in ewes previously treated with MPA and eCG.

Association between chronic Anaplasma marginale and Babesia spp. infection and hematological parameters of taurine heifers.

The aim of this study was to investigate the association between chronic Anaplasma marginale and Babesia spp. infection and hematological parameters of pregnant and non-pregnant taurine heifers. Blood samples from 94 females were collected on the first day (D-10) of timed artificial insemination (TAI) protocol and on pregnancy diagnosis (D+34). Hematological parameters were determined and compared between pregnant (PG) and non-pregnant (NPG) heifers, and within group at different sampling days. Real-time PCR (qPCR) was used to determine A. marginale and Babesia bovis infection, and for absolute quantification of Babesia spp. between PG and NPG groups. Correlation analysis was performed between the number of gDNA copies (CN) of Babesia spp. and hematological parameters. On D-10, mean hemoglobin concentration was higher for NPG, and hematocrit and total plasma protein were higher on D+34 for both groups. There was no difference in Babesia spp. CN between groups. In the first qPCR, all heifers were positive for A. marginale and B. bovis. Significant correlations were found between hemoglobin and erythrocyte and between hemoglobin and hematocrit (r = 0.8082 and r = 0.3009, respectively). Low levels of A. marginale and Babesia spp. did not affect hematological parameters of chronically infected pregnant and non-pregnant taurine heifers.

GnRH34 with or without estradiol cypionate in timed AI in Bos indicus beef cows.

Two experiments were performed to evaluate the effects of GnRH treatment on the fertility of suckled Nelore beef cows treated with an estradiol/progesterone (E2/P4)-based protocol for timed artificial insemination (TAI). Experiment 1 focused on determining the effects of estradiol cypionate (EC) on ovulation in TAI cows treated with GnRH 34 h after removal of the intravaginal P4 device (IPD). Suckled cows (n = 26) were treated with 2 mg estradiol benzoate (EB) and IPD containing 1 g P4. After 8 days, IPDs were removed, and all cows were treated with 150 µg of d-cloprostenol (prostaglandin F2 alpha analog) and 300 IU of equine chorionic gonadotropin (eCG), then separated into two treatment groups consisting of cows who received 1) saline 0.9% i.m. (GnRH34 group) or 2) 0.6 mg i.m. of EC (EC-GnRH34 group). On day 9 (05:00 p.m.), all cows were given GnRH (10.5 µg of buserelin acetate) i.m. No differences were observed between the groups (P > 0.05) in the time of ovulation after IPD removal or in the proportion of cows ovulating. Experiment 2 focused on determining the effects of GnRH34 along with or in the absence of EC on day 8 on pregnancy per AI (P/AI) in postpartum beef cows. Cows (n = 981) were treated similarly to those in Experiment 1, but an additional group, the EC-GnRH48 group, was included, in which cows received EC on day 8 whereas those that did not show estrus received GnRH at TAI. Thus, in this experiment, groups consisted of GnRH34 (n = 322), EC-GnRH34 (n = 335), and EC-GnRH48 (n = 324). A higher rate of estrus expression was observed in cows treated with EC following IPD removal (EC-GnRH34: 69%, EC-GnRH48: 64.8%) than in cows in the GnRH34 group (45.6%). No difference in P/AI was observed between the treatment groups (P = 0.45), but P/AI in cows in the EC-GnRH34 group (64.2%) tended to be greater (P = 0.1) than in cows in the GnRH34 group (58%). In summary, although ovulation synchrony did not differ among the groups, P/AI in cows treated with EC and GnRH 34 h after IPD removal tended to be greater than in cows treated solely with GnRH; this was most likely due to a shorter proestrus/estrus period, considering the lower proportion of cows that displayed estrus in the GnRH34 group. Finally, given that P/AI did not differ between the EC-GnRH34 and EC-GnRH48 groups, our results suggest that, for cows not displaying estrus, administration of EC at the time of IPD removal followed by treatment with GnRH 48 h afterward represents the most cost-efficient TAI strategy for South American Zebu-based beef operations.

Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation.

Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells.

Does combining estradiol cypionate and GnRH for ovulation induction in recipient cows increase pregnancy rate after timed embryo transfer?

Estradiol cypionate (EC) or GnRH have been widely used for ovulation induction in timed embryo transfer (TET). EC administration increases the proportion of cows that show estrus, whereas GnRH promotes more synchronized ovulations. The aim of the present study was to evaluate the potential beneficial effects of combining EC and GnRH in TET. In experiment 1, no difference was observed on serum progesterone concentrations on Day 6 and 13 after GnRH treatment between GnRH and EC+GnRH groups. In experiment 2, pregnancy per embryo transfer (P/ET) did not differ (p = 0.69) between GnRH (62.8%) and EC+GnRH (58.7%) groups. In conclusion, combining EC and GnRH for ovulation induction does not increase progesterone secretion and pregnancy rate after TET in cattle.

Paraoxonase 1 activity in the sperm-rich portion of boar ejaculates is positively associated with sperm quality.

Associations of the activity of the paraoxonase 1 (PON1) enzyme with boar sperm quality still needs to be characterized, since boar ejaculates present distinct portions with differences in sperm concentration and quality. This study evaluated PON1 activity in the serum, in the distinct portions of boar ejaculates and estimated correlations with sperm quality parameters. Ejaculates and blood samples were collected from six boars for three weeks (two per week per boar; n = 36). Serum and post-spermatic portion PON1 activities were positively correlated (P = 0.01) but were both uncorrelated with the PON1 activity in the sperm-rich portion and in the whole ejaculate (P > 0.05). Differences in PON1 activity among boars were only observed in the sperm-rich portion of the ejaculate (P < 0.05). The PON1 activity in the serum and in the post-spermatic portion was generally negatively correlated with parameters of spermatozoa kinetics (P < 0.05). In the sperm-rich portion, PON1 activity was positively correlated with sperm concentration (P < 0.0001), curvilinear distance and velocity (both P < 0.05) and DNA integrity (P < 0.05), but negatively correlated with straightness and linearity (P < 0.05). Thus, boar ejaculates with increased PON1 activity in the sperm-rich portion may present increased concentration and spermatozoa with acceptable curvilinear velocity and distance and DNA integrity, which suggests that PON1 activity may be a biomarker for potential fertility.

Increased ß-hydroxybutyrate (BHBA) concentration affect follicular growth in cattle.

Metabolic stress conditions caused by negative energy balance (NEB) have been associated with reduced fertility in cows. ß-hydroxybutyrate (BHBA) is the main circulating ketone body, which accumulates within follicular fluid. The aim of this study was to evaluate the effects of BHBA on follicle growth and on ovulatory mechanisms in cattle. At 72 h after intrafollicular injection, there was a decrease in follicular diameter in BHBA group compared to control (P = 0.02). Furthermore, follicle growth rate was reduced post-treatment with BHBA in comparison to the control group (P < 0.03). The BHBA intrafollicular injection in follicles ≥ 12 mm, however, did not affect E2 and P4 concentrations in the follicular fluid. In addition, the relative abundance of genes involved in the ovulatory cascade (ADAM 17, AREG, EREG, PTGS2), steroidogenesis (CYP19A1, 3BHSD, STAR), cellular stress (SOD1, CAT, GPX1, HSPA5, XBP1s, XBP1u, ATF4, ATF6), monocarboxylic acid transporters (SLC16A1, SLC16A7) and apoptosis (XIAP) was similar between groups. In conclusion, the results of this study indicate that the increase in intrafollicular concentrations of BHBA affects follicular growth, but it does not compromise the ovulatory cascade and cellular homeostasis in bovine granulosa cells.

The influence of prorenin/(pro)renin receptor on progesterone secretion by the bovine corpus luteum.

The aim of this study was to evaluate the role of prorenin/(pro)renin receptor activation on luteal progesterone (P4) secretion. Our hypothesis was that the nonproteolytic activation of (pro)renin receptor [P(RR)] is part of the regulatory mechanism responsible for corpus luteum (CL) function. In the first three experiments, prorenin was found to stimulate the production of P4, which is not inhibited by an angiotensin receptor antagonist (saralasin), but rather by a renin/prorenin inhibitor (aliskiren), a MAPK1/3 inhibitor (PD325901) or an EGFR inhibitor (AG1478), which are evidence of nonproteolytic activation of prorenin. Moreover, prorenin induced phosphorylation of MAPK1/3 in luteal cells. Following these in vitro experiments, a sequence of in vivo experiments was performed demonstrating that the intrafollicular injection of aliskiren in preovulatory follicles impaired P4 secretion in cows that ovulated. Furthermore, all profibrotic genes studied were present in the CL and TGFB1 and FN1 mRNA were upregulated from day 5-10 post-ovulation. During luteolysis, REN was downregulated at 48 h, whereas TGFB1 and SERPINE1 were dramatically upregulated in luteal tissue at 12 h after PGF. In summary, these data are evidence that nonproteolytic activation of (P)RR is involved in luteal function.

Bone morphogenetic protein 15 intrafollicular injection inhibits ovulation in cattle.

Oocyte-derived bone morphogenetic protein 15 (BMP15) is one of the main local regulators of ovarian physiology, but its role in the regulation of preovulatory follicles and ovulation is not well established. Therefore, this study was conceived to determine the effect of intrafollicular injection (IFI) of BMP15 on final follicular growth, ovulation and luteinization in cattle. Initially, it was observed that relative mRNA abundance of the BMP15 receptor BMPR1B in granulosa cells was regulated by GnRH treatment, and it was negatively correlated (R2 = 0.5; P < 0.001) to progesterone concentration in follicular fluid (FF) from preovulatory follicles. The IFI of recombinant human BMP15 tended to inhibit the growth of dominant follicles, as evidenced by an average increase of only 7.7% in the follicular diameter (from 8.8 mm to 9.1 mm) at 36 h post injection compared to 36.4% increase (from 8.9 mm to 14 mm) in the control group. Injection of BMP15 into preovulatory follicles (12-14 mm), simultaneously to im GnRH treatment, inhibited ovulation compared to control group, but did not prevent luteinization and progesterone production. Most of preovulatory follicles injected with BMP15 became luteinized cysts. Collectively, these findings indicate a suppressive role of BMP15 on later follicular development and ovulation in cattle, but not on luteogenesis and progesterone secretion.

Local regulation of antral follicle development and ovulation in monovulatory species.

The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals.

Blood perfusion and diameter of bovine corpus luteum as predictors of luteal function in early pregnancy.

A feasible and non-invasive luteal function assessment which enables timely intervention to improve progesterone (P4) support at early pregnancy is not well-established. The aim of this study was to determine the correlation among morphological and functional assessment methods of corpus luteum (CL) on Day 5 (D5) following timed-artificial insemination effect on luteal blood perfusion (LBP), CL diameter and serum P4 concentration. Beef heifers (n = 89) were synchronized and inseminated (D0). On D5, CL was scanned by colour-Doppler ultrasonography using an LBP score (0 = absent; 3 = high blood perfusion); CL diameter was obtained and blood was collected. Diameter of CL had a positive linear effect on P4 concentration (p < .001); and larger CL diameter increased the probability to become pregnant (p < .05; odds ratio =1.21). Heifers with a CL larger than 14.95 mm had a higher pregnancy rate than heifers with a 14.95 mm or smaller CL (p < .05). Animals with LBP 2 and 3 had larger CL when compared to scores 0 and 1 (p < .001). Scores 1, 2 and 3 can accurately estimate heifers with higher P4 (accuracy =0.79, 0.72 and 0.61 respectively). In conclusion, LBP on Day 5 post-TAI allows to estimate heifers with higher P4 and larger CL size, and a larger CL diameter was positively associated with pregnancy rate.

GnRH34: An alternative for increasing pregnancy in timed AI beef cows.

Two experiments evaluated the effects of gonadotropin releasing hormone (GnRH) treatment on fertility of suckled Nelore beef cows treated with an estradiol/progesterone (P4)-based protocol for timed artificial insemination (TAI). Experiment 1 was designed to determine the effect of GnRH administered 34 h after P4 insert removal (GnRH34) on time of ovulation. Suckled cows (n = 34) were treated with 2 mg estradiol benzoate (EB) and an intravaginal insert containing 1.9 g of P4. Eight days later, P4 inserts were removed, and all cows received 150 µg of d-cloprostenol (prostaglandin F2 alpha analogue), 300 IU of eCG, and 1 mg of estradiol cypionate (ECP). On Day 9 (05:00 p.m.), cows were randomly distributed, according to the diameter of the pre-ovulatory follicle, in two treatments: 1) GnRH (n = 17) cows that received 10.5 µg of buserelin acetate, or 2) no further treatment (control, n = 17). Cows treated with GnRH 34 h after P4 insert removal ovulated earlier (P = 0.02) than control cows (66 ± 0.0 and 77.2 ± 4.3 h). Experiment 2 was designed to determine the effect of GnRH34 on the fertility of suckled beef cows. Nelore cows (n = 506) were treated as Experiment 1. On Day 8, cows were painted in the sacrocaudal region to identify cows that displayed estrus. On Day 9 (05:00 p.m.), cows were randomized to receive same treatment as Experiment 1, control (n = 252), or GnRH (n = 254). All cows were TAI 48 h after P4 insert removal. At TAI, estrus was evaluated, and deemed to have occurred in cows without a tail-head chalk mark (>75% paint loss). Cows treated with GnRH 34 h after P4 insert removal had greater (P = 0.01) pregnancy per AI (P/AI) than cows that only received ECP (63.0% and 50.4%). No difference (P = 0.5) was observed in the proportion of cows that displayed estrus between treatments. Furthermore, cows that displayed estrus had greater (P < 0.01) P/AI than cows that did not. Treatment with GnRH, given at 34 h after P4 insert removal, increased (P < 0.05) P/AI in cows that did not show estrus at TAI. In summary, treatment with GnRH 34 h after P4 insert removal was associated with earlier ovulation and resulted in greater P/AI in suckled Nelore cows treated with an estradiol/P4-based protocol for TAI.

Growth hormone increases DNA damage in ovarian follicles and macrophage infiltration in the ovaries.

Evidence points to an important role of the growth hormone (GH) in the aging process and longevity. GH-deficient mice are smaller, live longer than normal littermates, and females have an increased ovarian reserve. The aim of the study was to evaluate the role of GH in the ovarian reserve by evaluating DNA damage, macrophage infiltration, and granulosa cell number in primordial and primary follicles. Experiment 1 used GH-deficient Ames dwarf mice (df/df, n = 12) and their normal littermates (N/df, n = 12), receiving GH or saline injections. Experiment 2 included transgenic mice overexpressing bovine GH (bGH) (n = 6) and normal mice (N, n = 6). DNA damage (anti-γH2AX) and macrophage counting (anti-CD68) were evaluated by immunofluorescence. Female df/df mice had lower γH2AX foci intensity in both oocytes and granulosa cells of primordial and primary follicles (p < 0.05), indicating fewer DNA double-strand breaks (DSBs). GH treatment increased DSBs in both df/df and N/df mice. Inversely, bGH mice had a higher quantity of DSBs in both oocytes and granulosa cells of primordial and primary follicles (p < 0.05). Df/df mice showed ovarian tissue with less macrophage infiltration than N/df mice (p < 0.05) and GH treatment increased macrophage infiltration (p < 0.05). In contrast, bGH mice had ovarian tissue with more macrophage infiltration compared to normal mice (p < 0.05). The current study shows that GH increases DNA DSBs in oocytes and granulosa cells and raises macrophage infiltration in the ovaries, pointing to the role of the GH/IGF-I axis in maintenance of oocyte DNA integrity and ovarian macrophage infiltration in mice.

Expression of steroidogenic enzymes and TGFß superfamily members in follicular cells of prepubertal gilts with distinct endocrine profiles.

Regulation of the transforming growth factor beta (TGFß) superfamily by gonadotrophins in swine follicular cells is not fully understood. This study evaluated the expression of steroidogenic enzymes and members of the TGFß superfamily in prepubertal gilts allocated to three treatments: 1200 IU eCG at D -3 (eCG); 1200 IU eCG at D -6 plus 500 IU hCG at D -3 (eCG + hCG); and the control, composed of untreated gilts. Blood samples and ovaries were collected at slaughter (D0) and follicular cells were recovered thereafter. Relative gene expression was determined by real-time PCR. Serum progesterone levels were greater in the eCG + hCG group compared with the other groups (P < 0.01). No differences were observed in the expression of BMP15, BMPR1A, BMPR2, FSHR, GDF9, LHCGR and TGFBR1 (P > 0.05). Gilts from the eCG group presented numerically greater mean expression of CYP11A1 mRNA than in the control group that approached statistical significance (P = 0.08) and greater expression of CYP19A1 than in both the eCG and the control groups (P < 0.05). Expression of BMPR1B was lower in the eCG + hCG treatment group compared with the control (P < 0.05). In conclusion, eCG treatment increased the relative expression of steroidogenic enzymes, whereas treatment with eCG + hCG increased serum progesterone levels. Although most of the evaluated TGFß members were not regulated after gonadotrophin treatment, the downregulation of BMPR1B observed after treatment with eCG + hCG and suggests a role in luteinization regulation.

Prostaglandin F2α regulation and function during ovulation and luteinization in cows.

Although prostaglandins are important in the ovulation process, a precise role for prostaglandin F2α (PGF) has not been elucidated. This study aimed to evaluate the regulation of PGF receptor mRNA (PTGFR) in granulosa cells and the local effect of PGF on ovulation and luteinization. In Experiment 1, using samples collected in vivo before (Day 2), during (Day 3) and after (Day 4) follicular deviation, expression of PTGFR in bovine granulosa cells was more abundant in the dominant follicle after deviation than in subordinates (P < 0.05). However, the expression of PTGFR was not regulated (P = 0.1) in preovulatory follicles at different time-points (0, 3, 6, 12 and 24 h) after ovulation induction with GnRH. In Experiment 2, to assess the role of systemic PGF treatment on luteinization and vascularization of preovulatory follicles, flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug, was used to inhibit endogenous prostaglandin synthesis. Cows with preovulatory follicles were induced to ovulate with GnRH (0 h) and allocated to three groups: Control, with no further treatment; FM, treated with 2.2 mg/kg FM im 17 h after GnRH treatment; and FM + PGF, treated with FM 17 h after GnRH, followed by 25 mg dinoprost tromethamine (PGF) 23 h after GnRH treatment. FM injection was able to reduce the concentration of PGF in the follicular fluid (FF) (P < 0.001). However, contrary to our hypothesis, color Doppler ultrasound evaluations revealed decreased vascular flow in FM + PGF group (P < 0.05), and no effect of the treatments on intrafollicular P4 and E2 concentrations 24 h after GnRH. The prostaglandin metabolite (PGFM) concentrations in the FF were greater in cows receiving systemic PGF (P < 0.001), which prompted us to further check its role on ovulation. Therefore, in Experiment 3, in a final attempt to demonstrate the local effect of PGF on ovulation, cows with preovulatory follicles received an intrafollicular injection (IFI) of PBS (Control) or 100 ng/mL purified PGF (PGF group). PGF treatment did not affect the time of ovulation after IFI (66 ± 6.4 and 63 ± 8.5 h for control and PGF, respectively; P > 0.05), further suggesting that it has no direct effect in the ovulatory process. Based on our findings, we concluded that FM decreased PGF synthesis within the follicle, whereas PGF treatment decreased follicular vascularization. In addition, the in vivo model of intrafollicular injection evidenced that PGF alone is not able to locally induce ovulation.

Regulation and function of leptin during ovarian follicular development in cows.

Although it is well documented that leptin signals the body nutritional status to the brain, mechanisms of leptin regulation at the ovary are not well understood. This study was conducted to determine whether there was leptin and the receptor for leptin (LEPR) in cattle ovarian follicles and to investigate potential actions of leptin on follicular growth in vivo and on regulation of granulosa cell functions in vitro. There was leptin and LEPR in granulosa and theca cells of dominant and subordinate follicles, with greater immunostaining for leptin in granulosa cells of subordinate follicles. There was a lesser relative abundance of leptin receptor gene-related protein (LEPROT) and of the adiponectin receptors 1 (ADIPOR1) and 2 (ADIPOR2) mRNA transcripts in granulosa cells of subordinate than dominant follicles (P < 0.05). Intrafollicular injection of either 100 or 1000 ng/mL leptin did not affect the diameter and the growth of dominant follicles (P> 0.05). Supplementation of in vitro culture medium with different leptin concentations did not affect (P > 0.05) the relative abundance of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), signal transducer and activator of transcription 3 (STAT3) and X-linked inhibitor of apoptosis protein (XIAP) mRNA transcripts in granulosa cells. These findings indicate that leptin and LEPR are present in the follicular cells of cattle ovaries, but leptin apparently does not have essential functions in steroidogenesis and growth of dominant follicles.

Primordial follicle reserve, DNA damage and macrophage infiltration in the ovaries of the long-living Ames dwarf mice.

The aim of this study was to evaluate the effect of growth hormone (GH) deficiency in primordial follicle reserve, DNA damage and macrophage infiltration in the ovaries of young mice. Ovaries from six-month-old GH-deficient Ames Dwarf (df/df) and Normal (N/df) mice were used. The number of primordial follicles was higher in df/df mice (p = 0.0026). Also, df/df mice had a lower number of primary (p = 0.023), secondary (p = 0.0052) and tertiary (p = 0.019) follicles. These findings indicate a slower rate of primordial follicle activation in df/df mice. Female df/df mice had decreased γH2AX foci intensity in oocytes of primordial (p = 0.015) and primary (p = 0.0004) follicles compared to N/df mice. Also, df/df mice had reduced γH2AX intensity in granulosa cells of primordial (p = 0.0002) and primary (p < 0.0001) follicles. Overall, this indicate to us that df/df mice accumulate less DNA damage in the ovarian reserve compared to N/df mice. Additionally, macrophage infiltration was also reduced in ovaries of df/df mice compared to N/df mice (p = 0.033). Interestingly, df/df mice had a reduced number of granulosa cells around primordial (p = 0.0024) and primary (p = 0.007) follicles compared to N/df mice. Also, df/df mice had a small diameter of primordial follicle nuclei (p = 0.0093), secondary follicle oocyte (p = 0.046) and tertiary follicle (p = 0.012). This points to the role of granulosa cell proliferation and oocyte growth for primordial follicle activation. The current study points to the role of the GH/IGF-I axis in extending lifespan of reproductive health, along with maintenance of oocyte DNA integrity and reduced ovarian inflammation.

Activation of PPARG inhibits dominant follicle development in cattle.

The peroxisome proliferator-activated receptor gamma (PPARG, also called NR1C3) is a nuclear receptor of the peroxisome proliferator-activated receptor family (PPAR). PPARs are involved in the regulation of apoptosis, cell cycle, estradiol and progesterone synthesis, and metabolism. However, the role of PPARs and their regulation during follicular development and ovulation in monovular species remain poorly understood. In this study, a well-established intrafollicular injection model was used to investigate if the PPARG participates in the regulation of dominant follicle development and ovulation in cattle. Findings from this study revealed that the relative mRNA abundance of PPARG was similar between dominant and subordinate follicles around follicle deviation, decreased after the LH surge, and increased before ovulation. In addition, a quadratic correlation was found between PPARG mRNA levels in granulosa cells and progesterone concentration in the follicular fluid. Intrafollicular injection of 50 µM Troglitazone (TGZ; a PPARG agonist) inhibited follicular growth and decreased CYP19A1 mRNA abundance in granulosa cells. These findings indicate that PPARG is involved in the regulation of steroidogenesis, follicle growth and ovulation in cattle.