Prostaglandin F2α treatment does not hasten ovulation in weaned sows.

This study evaluated the efficiency of prostaglandin F2α (PGF) to hasten ovulation in weaned sows. In experiment I, weaned sows detected in estrus (0 h) received: no hormone (Control; n = 56); 0.5 mg PGF IM at 0 h and 2 h (PGF0; n = 56); or 0.5 mg PGF IM at 24 h and 26 h (PGF24; n = 55). In experiment II, weaned sows that did not express estrus signs until 72 h after weaning (0 h) were assigned to: no hormone (Control; n = 45); 10 µg buserelin acetate IM at 0 h (Buserelin; n = 43); 0.5 mg PGF IM at 34 h and 36 h (PGF; n = 44); or 10 µg buserelin acetate IM at 0 h plus 0.5 mg PGF IM at 34 h and 36 h (Buserelin + PGF; n = 45). In experiment I, no effect of PGF on the interval treatment onset to ovulation was observed (P > 0.05), and no treatment effect was observed on the relative or cumulative proportion of females that ovulated post-treatment onset (P > 0.05). In experiment II, treatment onset to ovulation interval was shorter for Buserelin group than for PGF group (P < 0.05), and a higher cumulative percentage of Buserelin treated sows ovulated up to 48 h compared to PGF and Control groups (P < 0.01), with no differences from Buserelin + PGF. Treatments did not affect total number of piglets born in both experiments (P > 0.05). In conclusion, PGF did not hasten ovulation timing or affect litter size in weaned sows.

Long-acting injectable progesterone treatment prior to puberty induction in gilts.

Progesterone (P4) has a pivotal role on female puberty attainment in most farm animals. However, there are no studies evaluating the effect of P4 treatment previously to boar exposure for puberty induction in gilts. Therefore, serum P4 concentration, estrus expression and reproductive performance after boar stimuli were evaluated in gilts intramuscularly treated with long-acting P4 before boar exposure. In Experiment I, prepubertal gilts received either 1 mL of saline (control) or intramuscular (I.M.) P4 treatment (150 mg, 300 mg or 600 mg; n = 6 per treatment). Serum P4 concentration for P4-treated gilts was greater than for control gilts for at least 8 d for P4300 and P4600 groups (P < 0.05), but greater until after 16 d only for those treated with 600 mg (P < 0.05). In Experiments II (prepubertal) and III (peripubertal), gilts received either saline (control) or 300 mg P4 I.M. and those showing estrus signs were artificially inseminated (AI), whereas gilts without estrus expression were culled. In prepubertal gilts (Exp. II), estrus expression rate did not differ (P < 0.05) for control (79.1%; n = 110) and P4-treated gilts (81.5%; n = 108). In peripubertal gilts (Exp. III), although estrus expression did not differ between control (77.6%; n = 106) and P4-treated (69.6%; n = 102) gilts (P > 0.05), P4-treated gilts presented longer (23.1 ± 1.4 days) interval from treatment to estrus expression than control gilts (17.1 ± 1.3 days; P < 0.05). In Experiments II and III, the proportion of culled gilts with ovarian structures consistent with normal estrous cycles, farrowing rate, and litter size did not differ between treatments (P > 0.05). In conclusion, I.M. treatment with 300 or 600 mg of long-acting P4 was efficient in maintaining high P4 concentrations in prepubertal gilts for at least 8 days. However, P4 treatment over this time interval did not benefit the reproductive performance of prepubertal and peripubertal gilts.

Postponing TAI in beef cows with small preovulatory follicles.

The aims of this study were (1) to establish an optimal cut-off point for evaluating the effect of the preovulatory follicle (POF) diameter in timed AI (TAI) programs, and (2) to evaluate the effect of postponing TAI in cows with smaller follicles on pregnancy per AI (P/AI). In Study 1, Nelore cows (n = 426) were subjected to an estradiol-progesterone (P4) based TAI protocol. The diameter of POF was measured at TAI, 48 h after P4 insert removal. From the ROC curves for determining the relationship between the POF diameter and pregnancy, the cut-off was 11 mm (P < 0.001). Cows with larger POF had greater (P < 0.05) P/AI (62.5%) than cows with smaller POF (34.8%). In Study 2, Nelore cows (n = 1257) were subjected to the same TAI protocol as in Study 1. Before TAI, cows were separated according to POF diameter in three groups: (1) Larger POF (LP, n = 873; POF ≥11 mm), (2) Smaller POF 0 h (SP0, n = 195; POF <11 mm), and (3) Smaller POF 24 h (SP24, n = 189; POF <11 mm). Cows in the LP and SP0 were TAI at 48 h and cows in the SP24 group were TAI at 72 h after P4 insert removal. Cows in the LP group had the greatest (P < 0.05) P/AI (60.25%), followed by cows in the SP24 group (41.8%), while cows in the SP0 group had the lowest P/AI (31.8%). In conclusion, cows with smaller follicles have lower P/AI, but postponing the TAI by 24 h in these cows increases P/AI.

Pharmacological approaches to induce follicular growth and ovulation for fixed-time artificial insemination treatment regimens in ewes.

In Experiment I, during the non-breeding season, after intravaginal devices containing progesterone (P4) were withdrawn (n = 28), estrous rates were greater with treatment with 400 IU eCG (P < 0.05) than with FSH (10 and 15 mg) and no treatment. During the breeding season (n = 147), estrous and pregnancy rates after fixed-time artificial inseminations (FTAI) were similar among groups: 300 IU eCG; 10 mg FSH; and control (P > 0.05). In Experiment II (non-breeding season), ewes of one group were treated with 300 IU eCG (n = 8) and of two groups were treated with 10 mg FSH. In one FSH group, 250 µg estradiol benzoate (EB) were administered after 24 h (n = 9); in the other, 4 µg GnRH were administered after 36 h (n = 10). Serum P4 concentrations were greater in eCG-treated ewes (P < 0.05). Estrous rates were similar for the eCG- and FSH plus EB-treated ewes (P > 0.05). In Experiment III (breeding season), the treatments were: 300 IU eCG; 250 µg estradiol cypionate; 250 µg EB; or control (n = 22). Follicular growth was greater for eCG-treated ewes within 0-24 h and for control ewes within 48-72 h (P = 0.001). Although estrous and ovulation rates did not differ (P > 0.05), all eCG-treated ewes had ovulations. During the non-breeding season, FSH treatment promoted follicular growth but did not induce ovulations. For FTAI regimens, eCG was more effective than FSH plus GnRH and estradiol esters in inducing estrus and ovulation.

Induction of lactation in dairy heifers: milk production, inflammatory and metabolic aspects / Indução de lactação em novilhas leiteiras: produção de leite, aspectos inflamatórios e metabólicos

The aim of this study was to evaluate the metabolic, inflammatory, and hepatic aspects, as well as the milk yield in heifers submitted to protocol for induction of lactation compared to primiparous cows. Sixty Holstein heifers were selected and enrolled into two groups: Control (n= 30), pregnant heifers and Induction heifers (n= 30), non-pregnant femeales, submitted to a lactation induction protocol. Blood samples were collected at: pre-lactation period (weeks -3, -2 and -1) and post-lactation period (weeks 1, 2 and 3), aiming to evaluate glucose, non-esterified fatty acids, paraoxonase-1, albumin, ALT, GGT and cortisol. The protocol efficiently induced lactation in all the heifers, which produced 74.54% of the total production of milk from primiparous cows. In the pre-lactation period, induced animals presented higher concentrations of non-esterified fatty acids than the Control heifers, and the opposite was observed in the post lactation period. In both moments albumin and ALT were lower in the Induction group, and paraoxonase-1 activity and GGT concentrations were higher, compared to the Control. Thus, lactation induction protocol is efficient to initiate milk production in dairy heifers with no considerable changes in energetic, metabolic and hepatic profile when compared to heifers in physiological lactation.(AU)

O objetivo deste estudo foi avaliar os perfis metabólico, inflamatório, hepático e a produção de leite de novilhas induzidas à lactação comparadas a primíparas. Sessenta novilhas da raça Holandês foram selecionadas e alocadas em grupos: controle (n=30), novilhas prenhas, e indução (n=30), novilhas vazias submetidas a um protocolo de indução de lactação. As amostras de sangue foram coletadas nas semanas -3, -2 e -1 (pré-lactação) e nas semanas 1, 2 e 3 (pós-início de lactação) para avaliação de glicose, ácidos graxos não esterificados, paraoxonase-1, albumina, ALT, GGT e cortisol. O protocolo induziu eficientemente a lactação em todas as novilhas, que produziram 74,54% da produção total de leite do controle. No período pré-lactação, o grupo indução apresentou maiores concentrações de ácidos graxos não esterificados que o controle, e o oposto foi observado pós-lactação. Em ambos os momentos, albumina e ALT foram menores no grupo indução, e a atividade da paraoxonase-1 e as concentrações de GGT foram maiores, em comparação ao controle. Assim, o protocolo de indução de lactação foi eficiente para iniciar a produção de leite em novilhas induzidas, além de terem sido observadas alterações nos perfis energético, metabólico e hepático em comparação a novilhas em lactação fisiológica.(AU)

Treatment with eCG and hCG to induce onset of estrous cycles in ewes during the non-breeding season: Effects on follicular development and fertility.

Although combining of eCG and hCG administrations is known to enhance LH-like actions, there have been few studies where there was comparison of the effects of treatment of anestrous ewes with eCG and hCG and eCG alone. In Experiment 1, 18 ewes in seasonal anestrus were administered an intravaginal device (IVD) containing medroxyprogesterone acetate for 12 days, and at the time of IVD removal (D0), were allocated into the following groups (n = 6/group): no further treatment (control); 400 IU eCG (eCG); or 400 IU eCG and 200 IU hCG (eCG + hCG). There was greater ovarian follicular growth in the groups treated with gonadotropins, compared to the control, and there were greater progesterone concentrations in the eCG + hCG group on D9 (P < 0.05). In Experiment 2, 66 ewe lambs were assigned to the same treatment groups described for Experiment 1, and subsequently there was natural mating with rams. There was a greater rate of behavioral estrous manifestation in the eCG (88.5 %; 23/26) and eCG+hCG (85.2 %; 23/27), than control (30.8 %; 4/13; P < 0.05) group. Pregnancy rate was also greater in the eCG (34.6 %; 9/26) and eCG+hCG (18.6 %; 5/27) than control (0 %; 0/13; P < 0.05) group, whereas conception rate, considering only ewe lambs that were mated, was only greater in the eCG group. Although there were greater progesterone concentrations 9 days after treatment in the eCG+hCG group, there was no difference in follicular growth in anestrous ewes, nor was there an effect on estrous behavior manifestation and pregnancy rates in ewe lambs, compared to treatment with only eCG.

Effects of polyunsaturated fatty acids on the development of pig oocytes in vitro following parthenogenetic activation and on the lipid content of oocytes and embryos.

As oocytes and embryos of pigs have greater lipid content in the cytoplasm than those of other species, supplementation of the medium for in vitro maturation (IVM) of oocytes with omega-3 polyunsaturated fatty acids (PUFA) may help to improve embryo development. This study was conducted to evaluate effects of the inclusion of the docosaexaenoic (DHA) and of the eicosapentaenoic acids (EPA) in the IVM medium on the development of pig oocytes and on the lipid content of oocytes and embryos. In all experiments, control media consisted of porcine follicular fluid and oocytes were activated through parthenogenesis. In Experiment 1, there were four treatments for each PUFA: one control; and three treatments including EPA or DHA in the IVM medium at 12.5 µM, 25.0 µM and 50.0 µM). In Experiment 2, inclusion of 50 µM DHA was compared against the control. Cleavage rates in the IVM medium including 12.5 µM EPA and blastocyst development rates in media at any EPA concentration were less than for the control in Experiment 1 (P < 0.05). Compared to the control, inclusion of 50 µM DHA in the IVM medium was related to greater cleavage rates and greater number of embryo cells, in Experiment 1, and lesser lipid content in oocytes after 22 and 44 h and in embryos after 7 days, in Experiment 2 (both P < 0.05). Addition of DHA in the IVM medium may benefit the development of pig oocytes, but EPA appears to be cytotoxic.

Natriuretic peptide system regulation in granulosa cells during follicle deviation and ovulation in cattle.

Natriuretic peptides (NPs) are known to regulate reproductive events in polyovulatory species, but their function and regulation in monovulatory species remain to be fully characterized. Using a well-established in vivo model, we found that bovine granulosa cells from follicles near the deviation stage express mRNA for the three NP receptors (NPR1, NPR2 and NPR3), but not for NP precursors (NPPA, NPPB and NPPC). The abundance of NPR3 mRNA was higher in dominant compared to subordinate follicles at the expected time of follicular deviation. After deviation, mRNA for all NP receptors was significantly more abundant in the dominant follicle. Intrafollicular inhibition of oestrogen receptors downregulated NPR1 mRNA in dominant follicles. In granulosa cells from preovulatory follicles, NPPC mRNA increased at 3 and 6 h after systemic GnRH treatment, but decreased at 12 and 24 h to similar levels observed in samples collected at 0 h. After GnRH treatment, NPR1 mRNA was upregulated at 24 h, NPR3 mRNA gradually decreased after 3 h, while NPR2 mRNA was not regulated. The mRNA expression of the enzyme FURIN increased at 24 h after GnRH treatment. These findings revealed that the expression of mRNA encoding important components of the NP system is regulated in bovine granulosa cells during follicular deviation and in response to GnRH treatment, which suggests a role of NP system in the modulation of these processes in monovulatory species.

Expression of paraoxonase types 1, 2 and 3 in reproductive tissues and activity of paraoxonase type 1 in the serum and seminal plasma of bulls.

The paraoxonases types 1, 2 and 3 (PON1, PON2 and PON3, respectively) are enzymes that degrade lipid peroxides, preventing oxidative damages relevant for male reproductive function. This study determined the expression of those three paraoxonases in reproductive tissues of bulls and evaluated correlations among the activity of PON1 in the serum and seminal plasma with breeding soundness parameters in bulls. The expression of PON1, PON2 and PON3 was characterised by RT-PCR in samples of testicular parenchyma, vesicular glands and epididymis collected from three slaughtered bulls. All three paraoxonases were expressed in the testicular parenchyma, PON2 and PON3 were both expressed in the epididymis head and PON3 was also expressed in the epididymis tail. The PON1 activity was determined in samples of serum and seminal plasma from 110 bulls submitted to breeding soundness evaluation. There was a strong correlation (r = .90) between the activity of the PON1 in both serum and seminal plasma (p < .0001). The PON1 activity in the seminal plasma was positively correlated with ejaculate's colour, sperm mass activity (p = .04), motility, vigour and viability (all p < .01). Thus, PON1 may be a potential marker for sperm motility and viability in bulls.

Evidence that fibroblast growth factor 10 plays a role in follicle selection in cattle.

There is evidence that regulation of follicle selection in cattle involves locally produced growth factors. In the present study, we investigated the expression of members of the fibroblast growth factor (FGF) 7 family during follicle deviation. The largest and second largest follicles were recovered during the second day of a synchronised follicle wave and the future dominant and future subordinate follicles were identified based on diameter and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) mRNA levels in granulosa cells. Theca cells of the future dominant follicle contained less mRNA encoding FGF7 and FGF10 compared with those from the future subordinate follicle 2.5 days after ovulation, before a significant difference between the diameters of the future dominant and future subordinate follicles could be observed, but FGF22 mRNA levels did not change. Levels of mRNA encoding FGF receptors FGFR1B and FGFR2B in theca and granulosa cells, respectively, were lower in the future dominant follicle compared with the future subordinate follicle. Addition of FGF10 to granulosa cells in vitro significantly decreased oestradiol secretion, as well as CYP19A1, FSH receptor (FSHR) and insulin-like growth factor 1 receptor (IGF1R) mRNA abundance, whereas FGF22 had no effect. We conclude that FGF10 and FGFR2B expression is increased in the future subordinate follicle before morphological deviation, which may contribute to follicle selection.

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation.

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR-1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH-induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.

Metabolic and reproductive parameters in prepubertal gilts after omega-3 supplementation in the diet.

Polyunsaturated fatty acids may benefit reproductive performance of female swine. This study evaluated metabolic and reproductive parameters of prepubertal finishing gilts fed with fish oil as a natural source of omega-3 fatty acids (6.88g/d) (n=12) over a period of 45 d. Gilts in the control group were fed soybean oil (n=13). Body weight and backfat were determined at 15-d intervals. Serum levels of leptin, IGF-1, insulin, cholesterol and triglycerides were measured at the beginning (D0) and at the end of the period (D45). Immunolabeling intensity for leptin and its receptor (ObRb) was assessed in oocytes of preantral follicles. Gilts fed omega-3 presented slightly heavier uteri (P=0.09) than control gilts, but there was no effect on body weight and backfat (P>0.05). Cholesterol serum levels tended to be lower at D45 for omega-3 supplemented gilts than for controls (P=0.06). Triglycerides and IGF-1 serum levels were lower at D45 than at D0 for control gilts (P<0.05), but unaltered for supplemented gilts. Insulin levels were unaffected by supplementation (P>0.05), but were greater at D45 than at D0 in both treatments (P<0.05). Immunolabeling for leptin and ObRb in oocytes included in preantral follicles was more intense for supplemented gilts than for control gilts (P<0.05). Omega-3 supplementation was associated with reduced serum cholesterol level and more intense staining for leptin in oocytes of prepubertal gilts, which suggests some involvement on triggering puberty.

Regulation of Anti-Müllerian Hormone and Its Receptor Expression around Follicle Deviation in Cattle.

The anti-Müllerian hormone (AMH) is an important marker of ovarian reserve and for predicting the response to superovulatory treatments in several species. The objective of this study was to investigate whether AMH and its receptor (AMHR2) are regulated in bovine granulosa cells during follicular development. In the first experiment, granulosa cells were retrieved from the two largest follicles on days 2 (before), 3 (at the expected time) or 4 (after deviation) of follicular wave. In the second experiment, four doses of FSH (30, 30, 20 and 20 mg) or saline were administered twice a day starting on Day 2 of the first follicular wave of the cycle. Granulosa cells and follicular fluid were collected from the two largest follicles 12 h after the last injection of FSH or saline. AMH mRNA abundance was similar in granulosa cells of the two largest follicles (F1 and F2) before deviation (Day 2), but greater in dominant (DF) than subordinate follicles (SF) at the expected time (Day 3) and after (Day 4) deviation (p < 0.05). In experiment 1, AMH mRNA levels declined in both DF and SF near the expected time and after deviation when compared to before deviation. There was no difference in AMHR2 mRNA levels before and during follicular deviation (p > 0.05), but they tended to be greater in DFs than SFs (p < 0.1) after deviation. Experiment 2 showed that AMH and AMHR2 mRNA in granulosa cells and AMH protein abundance in follicular fluid were similar (p > 0.05) between both co-dominant follicles collected from the FSH-treated cows. These findings indicate the followings: AMH mRNA levels decrease in both DFs and SFs during follicular deviation; granulosa cells from heathy follicles express more AMH mRNA compared to subordinate follicles undergoing atresia and FSH stimulates AMH and AMHR2 mRNA expression in granulosa cells of co-dominant follicles.