Laboratory and Clinical Evaluation of a Feia Method for Canine Serum Progesterone Assay

The evaluation of progesterone (P4) concentration is a valuable tool in assessing physiological reproductive events and reproductive disorders in bitches. A reliable and rapid (preferable, point of care) determination of P4 is advisable in most cases. Aims of this study were to evaluate a fluorescence enzyme immunoassay (FEIA) for canine serum P4 concentration by (i) the agreement with liquid chromatography–tandem mass spectrometry (LC/MS/MS), (ii) the association with vaginal cytology and (iii) the accuracy in the prediction of the parturition date calculated from the estimated day of ovulation. Serum samples were collected from client‐owned bitches presented between 2011 and 2014 for the evaluation of their oestrous cycle, pregnancy or reproductive disorders. The agreement between FEIA and LC/MS/MS, evaluated on 19 samples, was statistically significant (R² = 95.7%, p < 0.001), although FEIA showed significantly higher values than LC/MS/MS (p < 0.05). In the different phases of oestrous cycle, as determined by vaginal cytology, P4 concentrations (by FEIA) were statistically different (p < 0.05): anoestrus (n = 7) 0.38 ± 0.14 ng/ml, proestrus (n = 14) 1.04 ± 0.67 ng/ml and oestrus (n = 72) 6.8 ± 7.26 ng/ml. Mean pregnancy length from the estimated day of ovulation was 62.9 ± 1.8 days. In 13 of 22 (59.1%), 19 of 22 (86.3%) and 21 of 22 (95.5%) bitches pregnancy lasted 63 ± 1, 63 ± 2 and 63 ± 3 days, respectively. Three pregnancies were outside the 61–65 days range (60, 60 and 67 days). In conclusion, the FEIA method employed can be considered reliable and, in association with vaginal cytology, effective in evaluating the canine oestrous cycle.     

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis,angiogenesis and survivability of cultured buffalo luteal cells

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P₄) secretion and mRNA expression of phosphotidylinositide‐3kinase‐protein kinase B (PI3K‐AKT), phosphoinositide‐dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P₄ secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P₄ secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P₄ secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.     

Ovarian and Hormonal Responses to Follicular Phase Administration of Investigational Metastin/Kisspeptin Analog,TAK‐683, in Goats

This study evaluated the effects of follicular phase administration of TAK‐683, an investigational metastin/kisspeptin analog, on follicular growth, ovulation, luteal function and reproductive hormones in goats. After confirmation of ovulation by transrectal ultrasonography (Day 0), PGF_2α (2 mg/head of dinoprost) was administered intramuscularly on Day 10 to induce luteal regression. At 12 h after PGF_2α administration, intravenous administration of vehicle or 35 nmol (50 μg)/head of TAK‐683 was performed in control (n = 4) and treatment (n = 4) groups, respectively. Blood samples were collected at 6‐h intervals for 96 h and then daily until the detection of subsequent ovulation (second ovulation). After the second ovulation, ultrasound examinations and blood sampling were performed every other day or daily until the subsequent ovulation (third ovulation). Mean concentrations of LH and FSH in the treatment group were significantly higher 6 h after TAK‐683 treatment than those in the control group (12.0 ± 10.7 vs 1.0 ± 0.7 ng/ml for LH, 47.5 ± 28.2 vs 15.1 ± 3.4 ng/ml for FSH, p < 0.05), whereas mean concentrations of oestradiol in the treatment group decreased immediately after treatment (p < 0.05) as compared with the control group. Ovulation tended to be delayed (n = 2) or occurred early (n = 1) in the treatment group as compared with the control group. For the second ovulation, ovulatory follicles in the treatment group were significantly smaller in maximal diameter than in the control group (3.8 ± 0.5 vs 5.4 ± 0.2 mm, p < 0.05, n = 3). Administration of TAK‐683 in the follicular phase stimulates gonadotropin secretion and may have resulted in ovulation of premature follicles in goats.     

Prolactin,Androstenedione and IGF1 Serum Concentrations During Induced Follicular Growth by eCG Administration in the Bitch

The oestrus cycle in the domestic bitch, a monoestrous species, differs considerably from that of other veterinary domestic animals species. In the bitch the combined use of eCG and hCG is effective to induce oestrus predictably and safely (Stornelli et al., Theriogenology, 78, 2012 and 1056). Although several studies were done to describe the hormonal changes during the canine oestrus cycle, to our knowledge none was done to describe the hormonal changes during induced follicular growth after the administration of eCG. The aim of this work was to study prolactin (PRL), insulin‐like growth factor (IGF1) and androstenedione (ANDR) serum concentrations during follicular growth induced by a single dose of eCG administered to late anoestrous bitches. PRL and ANDR concentrations were lower before than after eCG TRT (before eCG vs pro‐oestrus, oestrus and dioestrus; 4.3 ± 1.8 ng/ml vs 6.5 ± 1.6 ng/ml, p < 0.05; 0.08 ± 0.2 ng/ml vs 0.42 ± 0.16 ng/ml, p < 0.05). Conversely, IGF1 concentrations were similar before and after eCG TRT (286.0 ng/ml ±32.2, p > 0.53). Additionally, PRL concentrations were similar before oestrus compared to during oestrus and dioestrus (6.9 ± 1.7 ng/ml, p > 0.19). Furthermore, IGF1 concentrations were higher before and during oestrus compared to first day of dioestrus (286.1 ± 29.8vs 200.4 ± 29.2 ng/ml, p < 0.01). On the contrary, ANDR concentrations were lower before and during oestrus compared to first day of diestrum (0.35 ± 0.17 ng/ml and 0.38 ± 0.15 vs 0.68 ± 0.17 ng/ml, p < 0.05). These results show that treatment with a single injection of 50 IU/kg of eCG in late anoestrous bitches successfully induced changes in follicular growth which were paralleled with changes in PRL, IGF1 and ANDR serum concentration similar to those occurring during a normally occurring oestrous cycle. In addition, our results suggest that IGF1 in the bitch could play an important role in ovarian folliculogenesis.     

Endometrial Phospholipase A2 Activity During the Oestrous Cycle and Early Pregnancy in Mares

The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare’s endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1‐palmitoyl‐2‐[¹⁴C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μm . Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 thinsp;± thinsp;1.23 thinsp;nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity.     

Ion,Protein, Phospholipid and Energy Substrate Content of Oviduct Fluid During the Oestrous Cycle of Buffalo (Bubalus bubalis)

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre‐ovulatory, ovulatory, post‐ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post‐ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre‐ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (μm /24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post‐ovulatory phases (63.7 ± 11.2), with intermediate values in the pre‐ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre‐ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post‐ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (μmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre‐ovulatory and post‐ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species‐specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.     

Effect of Oestrus Synchronization with PGF2α/eCG/hCG on Luteal P4 Synthesis in Early Pregnant Gilts

Administration of hormones to synchronize oestrus is a useful tool in animal breeding. However, exogenous ovarian stimulation may be detrimental to reproductive function. This study was aimed to examine whether an oestrus synchronization with PGF2α/eCG/hCG could affect luteal P4 synthesis in early pregnant gilts. Corpora lutea (CLs) were collected on days 9, 12 and 16 of pregnancy from gilts with natural (n = 16) and synchronized (n = 18) oestrus and analysed for (i) the expre‐ssion of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A polypeptide (CYP11A1), and 3β‐hydroxysteroid dehydrogenase (3βHSD); (ii) the concentration of P4 in the luteal tissue and blood; and (iii) the expression of luteinizing hormone receptors (LHR) and oestrogen receptors (ERα and ERβ). Additionally, the effect of LH on P4 secretion from CL slices collected from synchronized and naturally ovulated animals has been studied in vitro. PGF_2α/eCG/hCG administration increased mRNA expression of StAR, CYP11A1, 3βHSD, and LHR on day 9 and CYP11A1 and LHR on day 12 of pregnancy compared with the control group (p < 0.05). CYP11A1, 3βHSD, LHR, ERα and ERβ proteins were not affected by synchronization; only StAR protein increased in hormonally treated animals (p = 0.017). The concentration of P4 in luteal tissue was greater on day 9 (p < 0.01), but lower on day 16 (p < 0.05) in gilts with hormonally induced oestrus compared with control animals. Blood serum levels of P4 were lower in synchronized than control gilts (p < 0.001). Synchronization did not affect LH‐stimulated P4 secretion from luteal slices; however, greater basal concentration of P4 in incubation medium was detected for CLs collected from synchronized than control gilts (p < 0.05). In conclusion, synchronization of oestrus with PGF2α/eCG/hCG protocol in gilts did not impair the expression of luteal P4 synthesis system, although decreased P4 concentration in the blood.     

Early resynchronization protocols for goats: Progestogens can be used prior to an early pregnancy diagnosis without affecting corpus luteum functionality

This study aimed to evaluate the exogenous progesterone (P4) effect on the luteal function from Day 16 to Day 21 of the oestrous cycle in inseminated goats with unknown pregnancy status. A total of 54 does passed through a short progestin-based synchronization protocol and, on Day 16 of the following oestrous cycle, 27 does received a new P4 device which was retained until Day 21. Blood samples were collected daily from all does during this period, as well as on Day 24. Pregnancy diagnoses were performed on Day 30. Serum P4 values from 26 animals (G_NPSP: Group of non-pregnant does with second sponge: n = 8; G_NPNSP: Group of non-pregnant does without second sponge: n = 6; G_PSP: Group of pregnant does with second sponge: n = 5; G_PNSP: Group of pregnant does without second sponge: n = 7) were determined by radioimmunoassay commercial kits. No P4 differences were found between groups (G_NPSP: 3.1 ± 2.8; 1.7 ± 1.8; 0.4 ± 1.0; and 0.0 ± 0.0 vs. G_NPNSP: 4.4 ± 1.8; 3.0 ± 2.2; 0.8 ± 0.8; and 0.0 ± 0.0 or G_PSP: 4.2 ± 1.0; 3.4 ± 0.6; 3.3 ± 1.6; 3.2 ± 0.9; 3.6 ± 1.2; 3.5 ± 1.3; 2.7 ± 1.3 vs. G_PNSP: 4.4 ± 1.6; 3.6 ± 1.5; 3.7 ± 1.5; 3.8 ± 1.4; 3.2 ± 1.2; 3.1 ± 1.2; 3.6 ± 1.1; D16, D17, D18, D19, D20, D21, D24, respectively) or for the interaction of group and time. In conclusion, a second progestogen device had no effect on luteolysis or early pregnancy in the following oestrous cycle.     

Expression of Heparin‐Binding EGF‐Like Growth Factor (HB‐EGF) in Bovine Endometrium: Effects of HB‐EGF and Interferon‐τ on Prostaglandin Production

Heparin‐binding EGF‐like growth factor (HB‐EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB‐EGF, prostaglandins (PGs) and interferon‐τ (IFN‐τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB‐EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB‐EGF and IFN‐τ on PGE2 and PGF2‐α production by endometrial cells were investigated. RT‐PCR analysis revealed that HB‐EGF mRNA was greater at the mid‐luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid‐ and late luteal stages than at the other luteal stages (p < 0.05). IFN‐τ increased the expression of HB‐EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB‐EGF did not affect PGF2‐α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2‐α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN‐τ significantly decreased HB‐EGF‐stimulated PGF2‐α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB‐EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN‐τ increased their expression in cultured endometrial cells. HB‐EGF and IFN‐τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.     

The post-ovulatory rise in progesterone is lower and the persistence of oestrous behaviour longer during the first compared with the second cycle of the breeding season in mares

Mares are seasonally polyoestrous breeders. Therefore, the first ovulation of the season, following winter anoestrus, is the only cycle in which mares ovulate without the presence of an old CL from the previous cycle. The objective of this study was to compare the length of oestrous behaviour, and plasma progesterone concentrations during the early post-ovulatory period between mares after the first and second ovulation of the breeding season. Overall, 38 mares and 167 oestrous periods were used in the study. From those, 11 mares were used during the first and subsequent oestrous period to measure and compare the post-ovulatory rise in progesterone concentration, whereas all the mares were used to compare the length of the post-ovulatory oestrous behaviour between the first and subsequent cycles of the breeding season. The persistence of the post-ovulatory oestrus was longer (p < .001) following the first ovulation of the year (median of 52 h) compared with the subsequent ovulations (median of 36 h for second and later ovulations groups; n = 38 mares). The progesterone concentration at any of the four 8 h-intervals analysed (28, 36, 76 and 84 h post-ovulation) was lower (p < .01) following the first versus the second ovulation of the year. By 36 h post-ovulation the progesterone concentration of mares at the second ovulation of the year had passed the threshold of 2 ng/ml (2.1 ± 0.33 ng/ml), whereas in the first cycle it was 1.2 ± 0.13 ng/ml. In conclusion, mares had lower progesterone concentrations in their peripheral circulation and longer persistence of oestrous behaviour following the first ovulation of the year compared with the second and subsequent ovulatory periods of the breeding season.     

Patterns of corpora lutea growth and progesterone secretion in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig)

The current study aimed to compare luteal function, as measured by corpora lutea dynamics and progesterone secretion, in 10 sows with obesity/leptin resistance genotype (Iberian pig) and 10 females of lean commercial crosses (Large White × Landrace). In all the animals, the oestrous cycle was synchronized with progestagens, and ovulation was induced by exogenous gonadotrophins. Thereafter, number and size of follicles and plasma oestradiol concentration were determined at oestrus detection, and number and size of corpora lutea and progesterone concentration were evaluated from Day 4 to 12 of the cycle. There were no differences between genotypes in follicle population and oestradiol concentration, and ovulation rate (15.2±1.3 in Iberian vs 12.7±1.8 in LWxL sows); however, there was a higher percentage of Iberian than control sows showing luteal cysts (66.7% vs 30%, respectively; p<0.05). In both breeds, both total luteal area and plasma progesterone concentration grew linearly from Day 4 to 8 (p<0.01) and remained more stable between Days 8 and 12, without significant differences between genotypes. In conclusion, current study supports that ovulatory processes and luteal functionality are not the main limiting factors for prolificacy in a pig model of leptin resistance and obesity.     

Influence of a bovine respiratory disease vaccine with a temperature‐sensitive modified live or killed infectious bovine rhinotracheitis component on oestrous cycle parameters and anti‐Müllerian hormone concentration in nulliparous heifers

The objective of this study was to examine the impact of a bovine respiratory disease complex (BRDC) vaccine with a temperature‐sensitive modified live vaccine (MLV) infectious bovine rhinotracheitis (IBR) component on oestrous cycle parameters and the follicular pool. Twenty‐four Holstein heifers (12.4 ± 0.5 months) previously calfhood vaccinated with an IBR MLV component were enrolled in two replicates (Spring; n = 10 and Fall; n = 14) and were blocked by pre‐vaccination bovine viral diarrhoea (BVD) serum neutralizing (SN) titres. Upon enrolment, heifers were oestrous synchronized with sampling beginning at detected oestrus. At their second heat, heifers were vaccinated with a BRDC calfhood vaccine with a MLV (MLV; n = 12) or killed (K; n = 12) IBR component and sampled for two additional cycles. Serum samples for oestrogen (E2) and progesterone (P4) as well as ultrasound data of ovarian structures were collected every other day. Serum samples for anti‐Müllerian hormone (AMH) were collected at oestrus and mid‐cycle for each cycle, and serum for titres was collected prior to and following vaccination. Data were analysed with the PROC MIXED and GLM procedures of SAS. There was no difference in pre‐ or post‐vaccination titres between MLV and K heifers (p > .5). Vaccination had no impact on P4 concentrations, P4 area under the curve, luteal tissue area, peak E2 production or oestrous cycle length (p > .05). Cycle number did impact AMH concentration (p < .05). In MLV heifers, AMH concentration was highest in cycle 1 (p < .05) while cycles 2 and 3 did not differ (p > .05). This was also true for the K heifers in the Fall replicate (p < .05). Within cycle 2, AMH concentrations were numerically lower between vaccine types (K = 308.22 ± 33.3 pg/ml, MLV = 181.13 ± 32.9 pg/ml; p > .05). Although no differences were seen in overall cycle parameters, differences in AMH concentrations may indicate a reduction of the follicular pool following vaccination and requires further investigation.     

Effect of eCG dose on ovarian haemodynamics,hormonal profiles and prolificacy rate when oestrus was induced during low-breeding season in Beetal goats

The objectives of the experiment were to determine the effect of two doses of equine chorionic gonadotropin (eCG) in a standard synchronization protocol based on a short-term progesterone (P₄) priming on ovarian structures and haemodynamics, concentrations of steroid hormones and prolificacy rate when oestrus was induced during low-breeding season (LBS) in Beetal dairy goats. We hypothesized that inclusion of eCG in a short-term P₄ priming-based synchronization protocol would increase the blood perfusion to ovarian structures leading to enhance oestrous and ovulatory responses and prolificacy rate in goats. Forty-two multiparous acyclic goats were blocked by body condition and, within block, assigned randomly to receive saline as control (CON), low eCG (L-eCG; 300 IU) or high eCG (H-eCG; 600 IU) dose. Initially, a controlled internal drug release (CIDR) device was placed in the anterior vagina on d −8, followed by removal of CIDR on d −3, concurrent with the administration of PGF_2α and eCG according to their respective treatments. Goats were monitored for oestrous response. B-mode and Doppler ultrasonography was performed with 12-h interval, starting from day −3 until natural breeding (day 0), and then on days 5, 10, 15 and 20 post-breeding to monitor follicular and luteal dynamics and blood flow, respectively. Blood was sampled at 0, 12, 24, 36 and 60 h after CIDR removal to quantify plasma concentrations of estradiol-17β (E₂), whereas plasma concentrations of P₄ were assayed at days 5, 10, 15 and 20 after breeding. Pregnancy and prolificacy rates were determined at day 30 and 150 after breeding, respectively. Data were analysed with mixed-effects models, and orthogonal contrasts were used to evaluate the effect of treatment [Con vs. (½ L-eCG + ½ H-eCG)] and dose of eCG (L-eCG vs. H-eCG). Data are presented in sequence as CON, L-eCG, H-eCG (LSM ± SEM). The oestrous intensity score (152.9 vs. 182.7 vs. 186.5 ± 15.1; p = .02) was greater in eCG-treated goats as compared to CON. Administration of eCG reduced the intervals to standing oestrus (66.2 vs. 41.8 vs. 48.9 h ± 5.5; p = .05), breeding (70.2 vs. 44.4 vs. 45.4 h ± 4.5; p = .03) and ovulation (84.5 vs. 61.2 vs. 63.4 h ± 6.2; p = .05) compared with CON goats. The mean growth rate of pre-ovulatory follicle was greater (1.11 vs. 1.49 vs. 1.45 mm ± 0.08; p = .01) in eCG-treated goats resulting in an increased diameter of pre-ovulatory follicle (6.27 vs. 7.20 vs. 7.31 mm ± 0.07; p < .01) and corpora lutea (6.75 vs. 8.26 vs. 8.07 mm ± 0.42; p = .04) than CON. The mean follicular blood flow did not differ among treatments; however, the mean luteal blood flow was greater in L-eCG-treated goats (0.81 vs. 1.61 vs. 1.07 cm² ± 0.12; p = .001). The mean concentrations of E₂ (4.03 vs. 5.21 vs. 4.78 pg/ml ± 0.42; p = .04) and P₄ (4.85 vs. 6.39 vs. 6.22 ng/ml ± 0.34; p = .04) were greater in eCG-treated goats. The twinning rate did not differ between treatments; nevertheless, prolificacy rate was greater (p = .04) in L-eCG-treated goats. Collectively, our data suggest that the administration of eCG improves the induction of oestrous and ovarian dynamics. Administration of L-eCG enhances prolificacy rate, therefore, a low dose of eCG might be practically beneficial to improve reproduction during LBS in acyclic Beetal dairy goats.     

Serum Levels of Cardiac Markers NT‐proANP and NT‐proBNP in Brachycephalic bitches at Different Gestational Stages

The aim of this study was to determine serum levels of natriuretic peptide precursors (NT‐proANP and NT‐proBNP) during pregnancy in brachycephalic bitches. Fifteen healthy multiparous bitches were selected for this prospective study. Serum levels of NT‐proANP and NT‐proBNP were measured during anoestrous and at 14, 35, 42, 49 and 56 days (2nd, 5th, 6th, 7th and 8th weeks) of pregnancy. Fourteen animals had normal gestations, and one bitch developed single foetus syndrome. The natriuretic peptide levels of this animal were not included in this study; however, it is important to report that its NT‐proANP levels were four times greater than those of normal patients. There was no significant difference (p = 0.072) in NT‐proBNP levels between anoestrous (0.20 ± 0.10 ng/ml) and the different pregnancy weeks (0.27 ± 0.12 ng/ml). There was a positive correlation (p < 0.0001) between NT‐proANP and gestational age, and the levels of this marker increased significantly (p < 0.0001) during the 6th (0.26 ± 0.06 ng/ml), 7th (0.28 ± 0.04 ng/ml) and 8th weeks (0.29 ± 0.05 ng/ml) when compared to anoestrous (0.18 ± 0.02 ng/ml). NT‐proANP serum levels are correlated with gestational development and may be indicative of cardiovascular adaptation in canine brachycephalic pregnancy.     

Anti‐Mullerian Hormone Concentration and Antral Ovarian Follicle Population in Murrah Heifers Compared to Holstein and Gyr Kept Under the Same Management

This study was performed to evaluate plasma concentrations of anti‐Mullerian hormone (AMH) and the ovarian antral follicle population (AFP) in different genetic groups. Cyclic heifers (13 Bubalus bubalis [Murrah]; 15 Bos taurus [Holstein] and 10 Bos indicus [Gyr]) were maintained under the same management and were synchronized with two doses of 150 μg IM d‐cloprostenol administered 14 days apart. After the second d‐cloprostenol treatment, heifers had their ovaries scanned daily by ultrasound to define the day of ovulation. On the same day, the AFP was determined and a plasma sample was collected to measure AMH. Murrah heifers had less AFP (25.6 ± 2.1 follicles; p = 0.01) and plasma AMH concentration (0.18 ± 0.03 ng/ml; p < 0.001) than Gyr (60.0 ± 12.2 follicles and 0.60 ± 0.12 ng/ml of AMH); however, data were similar when compared to Holstein (35.9 ± 6.8 follicles and 0.24 ± 0.06 ng/ml of AMH) heifers. Regardless of genetic background, there was a positive relationship between the AFP and plasmatic AMH concentration (Murrah [r = 0.62; p < 0.01], Holstein [r = 0.66; p < 0.001] and Gyr [r = 0.88; p < 0.001]). Also, when heifers were classified according to high‐ or low‐AMH concentration based on the average within each genetic group, high‐AMH heifers had greater (p < 0.0001) AFP than low‐AMH heifers. In conclusion, both Murrah and Holstein heifers presented lower plasma AMH concentration and AFP when compared to Gyr.     

Effects of L‐citrulline supplementation on heat stress physiology,lactation performance and subsequent reproductive performance of sows in summer

Lactating sows are susceptible to heat stress (HS). Part of the thermoregulatory response to HS is to increase peripheral blood flow, which is mediated in part by the vasodilator, nitric oxide (NO). Therefore, the aim of this experiment was to determine the effect of supplementation of L‐citrulline, a NO precursor, on symptoms of HS, lactation performance and subsequent reproductive performance of sows in summer. A total of 221 summer farrowing mixed parity sows were fed either a control diet or supplemented with 1% L‐citrulline upon entry to the farrowing house (6 ± 1.8 days for mean ± standard deviation [SD] before farrowing) until weaning (26 ± 1.5 days). The average daily minimum and maximum temperature in the farrowing house was 21.0 ± 1.88 and 29.2 ± 3.82°C (mean ± SD). Rectal temperature, respiration rate, and plasma and urinary nitrite and nitrate (NOx) of sows were measured on the 19th day post‐farrowing. Supplemental L‐citrulline in the diet did not affect the number of piglets born alive, feed intake of sows, body weight or backfat thickness of sows at weaning, or litter weight gain. L‐citrulline tended to reduce piglet pre‐weaning mortality rate from 18.6% to 15.6% (p = 0.058). L‐citrulline reduced the respiration rate of sows compared to the control diet at 17:00 hr (Time × Diet, p < 0.001); however, rectal temperature was not affected. L‐citrulline tended to increase urinary NOx concentrations (127 vs. 224 µM, p = 0.057) but not plasma NOx concentrations. L‐citrulline did not affect farrowing rate or number of piglets born alive in the subsequent parity. In conclusion, L‐citrulline supplementation reduced respiration rate of lactating sows and reduced piglet pre‐weaning mortality rate in summer. Whether the effects were due to a NO‐dependent mechanism requires further validation.     

Fertility of Sows Fed ad libitum with a High Fibre Diet During Pregnancy

The effect of ad libitum (ADLIB) feeding strategy on the fertility of the group housed sow was studied in a prospective field trial during 1.5 years. All study animals farrowed under standard farrowing circumstances in crates, and they were provided with an ad libitum feeding throughout the 30‐day lactation. After weaning, animals were randomly allocated to one of the two dry sow feeding strategies (AD LIB or CONT). After oestrus detection in groups, they were artificially inseminated and moved into pregnancy pens with partially slatted floor, in groups of 40 sows each. The ADLIB sows (n = 447) were loose housed and provided with ad libitum access to 7.7 MJ/kg feed high in fibre from two feeders per group. The control sows (n = 479; CONT) were also loose housed and given a standard dry sow feed in feeding stalls once a day (2.5 kg/day. The energy content of the feed was 9.3 MJ/kg NE). The feeding strategy (ADLIB vs CONT) had no effect on pregnancy rate (85.8 vs 90.9, p > 0.05), weaning to oestrus interval (7.7 vs 7.3 days, p > 0.05), piglets born alive (11.5 ± 3.5 vs 11.6 ± 3.3, p > 0.05), stillborn piglets (1.2 ± 1.8 vs 0.9 ± 1.5, p > 0.05) nor on progesterone concentration (p > 0.05). CONT sows weaned more piglets (9.7 ± 2.2 vs 9.4 ± 2.0, p < 0.01), whereas the piglets of AD LIB sows were heavier at weaning (8.8 ± 0.9 vs 8.0 ± 1.3 kg, p < 0.05). In conclusion, ad libitum feeding with a high in fibre diet during pregnancy did not affect the reproductive performance.     

Effects of Dietary Supplementation of β‐hydroxy‐β‐methylbutyrate on Sow Performance and mRNA Expression of Myogenic Markers in Skeletal Muscle of Neonatal Piglets

The effects of dietary β‐hydroxy‐β‐methylbutyrate (HMB) supplementation during gestation on reproductive performance of sows and the mRNA expression of myogenic markers in skeletal muscle of neonatal pigs were determined. At day 35 of gestation, a total of 20 sows (Landrace × Yorkshire, at third parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 4 g/day β‐hydroxy‐β‐methylbutyrate calcium (HMB‐Ca) until parturition. At parturition, the total and live litter size were not markedly different between treatments, however, the sows fed HMB diet had a decreased rate of stillborn piglets compared with the sows fed the control (CON) diets (p < 0.05). In addition, piglets from the sows fed HMB diet tended to have an increased birth weight (p = 0.08), and a reduced rate of low birth weight piglets (p = 0.05) compared with piglets from the CON sows. Nevertheless, lower feed intake during lactation was observed in the sows fed the HMB diet compared with those on the CON diet (p < 0.01). The relative weights of the longissimus dorsi (LD) and semitendinosus (ST) muscle were higher (p < 0.05) in neonatal pigs from the HMB than the CON sows. Furthermore, maternal HMB treatment increased the mRNA levels of the myogenic genes, including muscle regulatory factor‐4 (MRF4, p < 0.05), myogenic differentiation factor (MyoD) and insulin‐like growth factor‐1 (IGF‐1, p < 0.01). In conclusion, dietary HMB supplementation to sows at 4 g/day from day 35 of gestation to term significantly improves pregnancy outcomes and increases the expression of myogenic genes in skeletal muscle of neonatal piglets, but reduces feed intake of sows during lactation.     

Evaluation of haemodynamic changes of uterine arteries using Doppler ultrasonography during different stages of pregnancy in Bos indicus cows

The objectives of the study were to evaluate haemodynamic changes and their relationships among ipsilateral (IPS) and contralateral (CONT) uterine arteries (UA) during different stages of pregnancy in Bos indicus cows. Multiparous pregnant cows (n = 40) having a gestation length 30.47 ± 0.54 (mean ± SD) days were randomly enrolled and subjected to Doppler ultrasonography sequentially at 1st, 2nd, 4th, 6th and 8th months of gestation. Blood flow indices including diameter of UA (mm), blood flow volume (BFVo, ml/min), blood flow velocity (BFVe, cm/s), time-averaged maximum velocity (TAMV, cm/s), pulsatility index (PI) and resistance index (RI) were recorded. Data were analysed with mixed models using the PROC MIXED procedures, and Pearson correlation coefficients were calculated using the PROC CORR statement in SAS. The final statistical models included the fixed effects of side of UA, gestation month and the interaction between side of UA and gestation month. Results revealed that the mean diameter of the UA (12.13 ± 0.22 vs. 10.09 ± 0.22), BFVo (1236.33 ± 0.55 vs. 770.41 ± 0.55), BFVe (17.18 ± 0.42 vs. 15.58 ± 0.42) and TAMV (17.11 ± 0.44 vs. 15.77 ± 0.44) was higher (p < .05) in IPS as compared to CONT side of the UA in cows. However, PI and RI did not differ between IPS and CON arteries of uterus in cows. A very high and positive correlation (r = .89; p < .05) existed between the diameter of UA and BFVo starting from 1st to 8th months of gestation in IPS as well as CONT sides of UA. Moreover, TAMV was highly and positively correlated (r = .91; p < .05) with BFVe throughout the gestation. In conclusion, these haemodynamic changes in the UA could be used as a valuable validity tool to differentiate the compromised pregnancy in Bos indicus cows.