Follicular Dynamics and Oestrous Detection in Thai Postpartum Swamp Buffaloes (Bubalus bubalis)

This study characterized follicular activity and oestrous behaviour from 5 to 9 days post‐calving up to the 4th ovulation postpartum (pp) in 16 multiparous (range 2–7 parities) Thai swamp buffalo cows (Bubalus bubalis), aged 4–12 years and weighing from 432 to 676 kg. Ovarian follicular activity was examined by transrectal ultrasonography (TUS) every morning. Oestrous detection was performed twice daily by direct personal observation of behaviour and for presence of clear cervical mucus discharge and indirectly by video camera recording during 21 h/day. A follicular wave‐like pattern was present before the 1st ovulation leading to short oestrous cycles. Growth rates and maximum diameters of the ovulatory follicles did not differ between the 1st and 4th ovulations. However, growth rate for non‐ovulatory dominant follicles (DF) before the 1st ovulation was lower than for the ovulatory follicle (p < 0.05). In addition, the diameter of all ovulatory follicles (14.3 ± 0.46 mm, n = 39) was significantly larger (p < 0.01) than those of the preceding last but one non‐ovulatory DF (10.8 ± 0.20 mm, n = 5), but similar to the last preceding non‐ovulatory DF diameter (12.92 ± 0.96 mm, n = 14). Short oestrous cycles were most common between the 1st and 2nd ovulations (93.75%, 15/16 cows, 10.2 ± 0.38 days) decreasing in prevalence thereafter (50%, 3/6 buffaloes, 12.0 ± 1.53 days). Oestrous signs were relatively vague around the 1st ovulation pp to become more easily detectable thereafter. This study suggests that properly fed swamp buffaloes could be mated successfully within 2 months pp, at their 2nd spontaneous ovulation, provided oestrous detection is at least performed daily at 06:00–08:00 hour.     

Changes of Connexin43 Expression in Non‐pregnant Porcine Myometrium Correlate with Progesterone Concentration During Oestrous Cycle

Connexin43 (Cx43) is a major protein of myometrial gap junctions. The number of Cx43 gap junctions increase dramatically with the onset of labour in association with development of synchronized uterine contractions. The formation of myometrial gap junctions follows an increase in the oestrogen to progesterone ratio indicating an important role of steroid hormones in regulating Cx43 expression at term. However, no relationship has been established between the expression of Cx43 in the non‐pregnant myometrium and concentration of steroid hormones during the oestrous cycle. Here, we used immunofluorescence and Western blotting to analyse the expression of Cx43 gap junctions in the myometrium of pre‐pubertal pigs (n = 7) and mature pigs at pre‐ovulatory (n = 7), luteal (n = 5) and late luteal (n = 3) stages of the oestrous cycle. The number of Cx43 gap junctions calculated per 1 mm² of the myometrial section was low in pre‐pubertal pigs and significantly higher (p < 0.022) in pre‐ovulatory animals. In relation to pre‐ovulatory animals the number of myometrial gap junctions was significantly lower (p < 0.019) at the luteal phase and correlated with significantly higher (p < 0.005) concentration of endogenous progesterone. Phosphorylated isoforms of Cx43 protein were expressed in the myometrium of pre‐pubertal pigs and mature animals at pre‐ovulatory and late luteal phases, while they were down regulated at the luteal stage. These results indicate that changes of Cx43 expression in the porcine myometrium during the oestrous cycle may be regulated by progesterone concentration and may contribute to the modulation of uterine motility.     

Detection of Glycosylated Proteins in Rabbit Oviductal Isthmus and Uterine Endometrium During Early Embryo Development

In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of oviductal isthmus and uterine endometrium in rabbits during early embryo development. Rabbit embryos are surrounded not only by the zona pellucida but also by tubal secretion‐derived mucinous coat material, the mucin coat. Twenty sexually mature females were euthanized at 0 (pre‐ovulatory group) and 24, 72 and 96 h after insemination (pseudopregnancy group). The following lectin‐binding agents were used: Arachis hypogaea, Peanut (PNA) to label galactosyl (β‐1,3)N‐ acetyl‐galactosamine, Dolichos biflorus Agglutinin (DBA) to label galactosyl (β‐1,4)N‐ acetyl‐galactosamine, Lens curinaris (LCA) to label α‐‐mannose, α‐d ‐glucose and Pisum sativum agglutinin (PSA) to label α‐d ‐mannose, α‐d ‐glucose. Blood was collected by cardiac puncture, and direct enzyme immunoassay technique was used to measure progesterone concentration. A significant increase in total plasma progesterone concentrations was detected at 96 h post‐ovulation when compared with 0, 24 and 72 h post‐ovulation (2.9 ± 0.5 vs 0.5 ± 0.15, 1.6 ± 0.5 and 1.5 ± 0.4 ng/ml, at 96 h vs 0, 24 and 72 h post‐ovulation, respectively). No differences between pre‐ovulatory and pseudopregnant females were observed for glycoprotein localization in isthmus. In contrast, in the endometrium, differences in the glycoprotein detection between pre‐ovulatory and pseudopregnant stages were detected. PNA to label galactosyl (β‐1,3)N‐ acetyl‐galactosamine was not detected at the pre‐ovulatory stage, but its presence was detected at 24 h after ovulation. Both PSA and LCA to label α‐d ‐mannose, α‐d ‐glucose were only detected at 72 h after ovulation. DBA detection was similar for all stages of the reproductive cycle. Therefore, N‐acetyl‐galactosamine secreted from isthmus could be involved in the formation of the embryonic mucin coat. d ‐galactose (PNA), d ‐glucose and d ‐mannose (PSA and LCA) might be crucial for the implantation period.     

Induction of Ovulation of Ovulatory Size Non‐Ovulatory Follicles and Initiation of Ovarian Cyclicity in Summer Anoestrous Buffalo Heifers (Bubalus bubalis) Using Melatonin Implants

This study was conducted on summer anoestrous buffalo heifers to monitor the efficacy of melatonin for induction of ovulation and ovarian cyclicity. During pre‐treatment period of 24 days, the ovarian dynamics of five cycling and 10 summer anoestrous heifers was monitored on each alternate day using a transrectal ultrasound scanner. Thereafter, during treatment period, these 10 anoestrous heifers along with additional seven anoestrous heifers were randomly allocated into non‐implanted (n = 5) and implanted (n = 12, one melatonin implant/50 kg, 18 mg melatonin/implant) group. Non‐implanted heifers were monitored on each alternate day till the confirmation of second‐ovulation in implanted heifers. Pre‐treatment period revealed the presence of dominant follicles in anoestrous heifers which attained the diameter comparable with ovulatory follicles of cycling heifers but failed to ovulate and regressed. Between 6 and 36 days (15.3 ± 2.9 days) post‐treatment, all the implanted heifers (p < 0.05) exhibited ovulation of dominant follicles; however none of the non‐implanted heifers ovulated during the corresponding period. The first‐interovulatory period in implanted heifers ranged between 8 and 28 days (18.0 ± 1.8 days). The implanted heifers with short (≤16 days) interovulatory period had short‐lived corpus luteum (CL) that had smaller diameter and secreted less progesterone (p < 0.05). The diameter of CL was large (p < 0.05) and plasma progesterone was high (p < 0.05) following second‐ovulation compared with first‐ovulation in implanted heifers. In conclusion, using melatonin implants, ovulatory size nonovulatory follicles observed in summer anoestrous buffalo heifers can be successfully ovulated to initiate ovarian cyclicity.     

Relationship between Ultrasonographic Assessment of the Corpus Luteum and Plasma Progesterone Concentration during the Oestrous Cycle in Monovular Ewes

Ultrasonographic observations of the corpus luteum (CL) and collection of blood samples for progesterone radioimmunoassay were performed daily during 15 oestrous cycles in Spanish Merino ewes, a consistently monovular breed. Ultrasonographic image of the CL changed during the oestrous cycle, increasing its echogenic pattern from ovulation to luteolysis. The size of the CL and mean progesterone levels were significantly affected by day of cycle (p < 0.05 and p < 0.001, respectively). Both increased their values from day 1 to day 12 (from 49.6 ± 7.4 to 154.6 ± 11.8 mm² and from 0.2 ± 0.0 to 2.8 ± 0.5 ng/ml, respectively) and then declined sharply until day 0 (28.2 ± 5.3 mm² and 0.1 ± 0.0 ng/ml, respectively). There was a significant correlation between CL area and plasma progesterone concentrations during the entire oestrous cycle, taking the developing and regressing phases of the CL separately (p < 0.05). A central cavity was observed in 33.3% of the CL studied. The presence of this cavity had no effect in total luteal‐tissue area of the CL nor on oestrous cycle length or on progesterone concentrations. Likewise, the cavity did not affect the correlations observed between CL size and progesterone levels, CL size and day of cycle and progesterone levels and day of cycle. It is concluded that ultrasonographic assessment of CL area is a reliable method for estimating peripheral plasma progesterone levels, regardless to the presence or absence of a cavity in the CL.     

Endometrial glycogen, protein, nucleic acids and phosphatases during oestrous cycle in buffaloes (Bubalus bubalis).

Distinct cyclic variations were observed in the concentrations of endometrial glycogen, protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) during the follicular and luteal phases of the oestrous cycle in buffaloes. Glycogen, protein, DNA and RNA concentrations were significantly (P less than 0.01) higher in buffalo endometrium during the follicular phase than the luteal phase of the oestrous cycle whereas ACP and AKP activities were significantly (P less than 0.01) elevated during the luteal phase compared with the follicular phase. These results are discussed in relation to the nourishment of the blastocyst during the pre-implantation period.     

Luteal function and follicular growth following follicular aspiration during the peri-luteolysis period in Bos indicus and crossbred cattle

Follicular estradiol triggers luteolysis in cattle. Therefore, the control of follicle growth and steroidogenesis is expected to modulate luteal function and might be used as an anti‐luteolytic strategy to improve embryo survival. Objectives were to evaluate follicular dynamics, plasma concentrations of estradiol and luteal lifespan in Bos indicus and crossbred cows subjected to sequential follicular aspirations. From D13 to D25 of a synchronized cycle (ovulation = D1), Nelore or crossbred, non‐pregnant and non‐lactating cows were submitted to daily ultrasound‐guided aspiration of follicles >6 mm (n = 10) or to sham aspirations (n = 8). Diameter of the largest follicle on the day of luteolysis (7.4 ± 1.0 vs 9.7 ± 1.0 mm; mean ± SEM), number of days in which follicles >6 mm were present (2.3 ± 0.4 vs 4.6 ± 0.5 days) and daily mean diameter of the largest follicle between D15 and D19 (6.4 ± 0.2 vs 8.5 ± 0.3 mm) were smaller (p < 0.01) in the aspirated group compared with the control group, respectively. Aspiration tended to reduce (p < 0.10) plasma estradiol concentrations between D18 and D20 (2.95 ± 0.54 vs 4.30 ± 0.55 pg/ml). The luteal lifespan was similar (p > 0.10) between the groups (19.6 ± 0.4 days), whereas the oestrous cycle was longer (p < 0.01) in the aspirated group (31.4 ± 1.2 vs 21.2 ± 1.3 days). Hyperechogenic structures were present at the sites of aspiration and were associated with increase in concentration of progesterone between luteolysis and oestrus. It is concluded that follicular aspiration extended the oestrous cycle and decreased the average follicular diameter on the peri‐luteolysis period but failed to delay luteolysis.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

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.     

Effect of Oestrous Synchronization with Estradiol 17β and Progesterone on Follicular Wave Dynamics in Dairy Heifers

An experiment was designed to evaluate the effects of estradiol‐17β (E17β) on follicular wave dynamics and ovulatory response in Holstein heifers receiving either a progestogen ear‐implant (Crestar^®; Intervet International b.v. Boxmeer, The Netherlands) or an intravaginal progesterone‐releasing device [controlled internal drug release‐bovine device (Eazibreed, CIDR‐B^®; Bodinco BV, Alkmaar, The Netherlands)]. For comparison, another group of heifers was also synchronized using Crestar plus an injection of estradiol valerate (EV) and norgestomet as recommended by the pharmaceutical company. Twenty 20–22‐month‐old cycling Holstein heifers were allocated to one of the following treatment groups at random stages of the oestrous cycle: (I) simultaneous insertion of Crestar and intramuscular injection of 3 mg norgestomet and 5 mg EV (Crestar 9 + EV 9); (II) simultaneous insertion of Crestar and intramuscular injection of 5 mg E17β (Crestar 9 + E17β 9); (III) insertion of Crestar followed 2 days later by intramuscular injection of 5 mg E17β (Crestar 9 + E17β 7); or (IV) insertion of CIDR‐B device followed 2 days later by intramuscular injection of 5 mg E17β (CIDR 9 + E17β 7). The CIDR‐B or Crestar implants were removed after 9 days and all heifers received 500 μg Cloprostenol (Estrumate^®, Pitman‐Moore Nederland BV, Houten, The Netherlands). Ovarian ultrasonographic examinations were performed once daily during the synchronization period using a B‐mode scanner equipped with a 7.5 MHz linear‐array transrectal transducer. In addition, heifers were scanned every 12 h after implant/device withdrawal until 3 days after ovulation in order to monitor follicular activity, detect ovulation and subsequent early luteal formation. Detection of oestrus was performed every 6 h for 4 days after device/implant removal. Oestrus was observed 24–32 h before ovulation in all heifers. The mean hours interval from treatment withdrawal to ovulation was not significantly different (84.0 ± 16.5, 77.6 ± 4.1, 73.6 ± 4.1 and 64.0 ± 4.4 h for treatments I, II, III and IV, respectively; p > 0.1). However, the variance for heifers treated with EV + norgestomet was significantly larger (Levene’s Test; p < 0.01) than those treated with E17β. All E17β treatments resulted in dominant follicle suppression and a new wave emerged 4.1 days after treatment compared with 6.6 days for the EV + norgestomet treatment (p < 0.05). The time from emergence of the new ovulatory wave to ovulation was longer for the new wave that emerged after E17β treatment (9.2 ± 0.3 days) than after EV + norgestomet treatment (6.9 ± 0.4 days; p < 0.05). The results of this study suggest that the four treatments used were effective in inducing synchronous behavioural oestrus and ovulation. However, a higher degree of oestrus and ovulation synchrony was observed in heifers treated with E17β than in heifers treated with EV + norgestomet. Synchronization treatments with exogenous E17β or EV + norgestomet at the time of progestin device insertion (Crestar or CIDR‐B) or 2 days later in heifers can regulate a different emergence pattern of ovarian follicular development in randomly cyclic heifers. The E17β was effective in inducing follicular suppression and resulted in the consistent emergence of a new follicular wave.     

Comparison of Different Treatments for Oestrous Induction in Seasonally Anovulatory Mares

The aim of this study was to evaluate the effects of different treatments for induction and synchronization of oestrus and ovulation in seasonally anovulatory mares. Fifteen mares formed the control group (C), while 26 mares were randomly assigned to three treatment groups. Group T1 (n = 11) were treated with oral altrenogest (0.044 mg/kg; Regumate^®) during 11 days. Group T2 (n = 7) was intravaginally treated with 1.38 g of progesterone (CIDR^®) for 11 days. In group T3 (n = 8), mares were also treated with CIDR^®, but only for 8 days. All mares received PGF2α 1 day after finishing the treatment. Sonographic evaluation of follicles, pre‐ovulatory follicle size and ovulation time was recorded. Progesterone and leptin levels were analysed. Results show that pre‐ovulatory follicles were developed after the treatment in 88.5% of mares. However, the pre‐ovulatory follicle growth was dispersal, and sometimes it was detected when treatment was not finished. While in mares treated with intravaginal device, the follicle was soon detected (1.5 ± 1.2 days and 2.3 ± 2.0 days in T2 and T3 groups, respectively), in T1 group, the pre‐ovulatory follicle was detected slightly later (3.9 ± 1.6 days). The interval from the end of treatment to ovulation did not show significant differences between groups (T1 = 13.1 ± 2.5 days; T2 = 11.0 ± 3.6 days; T3 = 13.8 ± 4.3 days). The pregnancy rate was 47.4%, similar to the rate observed in group C (46.7%; p > 0.05). Initial leptin concentrations were significantly higher in mares, which restart their ovarian activity after treatments, suggesting a role in the reproduction mechanisms in mares. It could be concluded that the used treatments may be effective for oestrous induction in mares during the late phase of the seasonally anovulatory period. Furthermore, they cannot synchronize oestrus, and then, it is necessary to know the reproductive status of mares when these treatments are used for oestrous synchronization.     

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.     

The Content of β-endorphin-like Immunoreactivity in Porcine Corpus Luteum and the Potential Roles of Progesterone, Oxytocin and Prolactin in the Regulation of β-endorphin Release from Luteal Cells in vitro

The amount of β‐endorphin‐like immunoreactivity (β‐END‐LI) in porcine corpora lutea from several stages of the oestrous cycle and the effects of progesterone, oxytocin, and prolactin on β‐END‐LI secretion in vitro by luteal cells were studied. Porcine corpora lutea obtained on days 1–5, 6–10, 11–13, 14–18, and 19–21 of the cycle were used to prepare extracts for β‐END‐LI determination. Additionally, corpora lutea from days 11–13 and 14–18 were enzymatically dissociated and isolated luteal cells were used for further study of β‐endorphin secretion in vitro. Cells were cultured in serum‐free defined M 199 medium (10⁶ cells/ml) at 37°C under 5% CO₂ in air, for 12 h. The influences of the following factors on β‐END‐LI secretion by luteal cells were tested: progesterone (10^–9, 10^–7 and 10^–5M ), oxytocin (0.01, 0.1, 1 and 10 ng/ml), and prolactin (0.1, 1, 10 and 100 ng/ml). The β‐END‐LI contents in extracts and media were measured by radioimmunoassay. The tissue concentration of β‐END‐LI was lowest on days 1–5 of the cycle (0.35 ± 0.03 ng/g wet tissue). Subsequently, it constantly increased to the highest value on days 14–18 (16.58 ± 0.52 ng/g wet tissue) and on days 19–21 it declined (11.10 ± 0.52 ng/g wet tissue). Progesterone at a low dose (10^–9 M ) resulted in significant (p < 0.05) increases and decreases in β‐END‐LI secretion by luteal cells from days 11–13 and 14–18, respectively. Higher doses of progesterone (10^–7 and 10^–5 M ) had no effect on β‐END‐LI release, compared with the control group. All dose‐levels of oxytocin used decreased β‐END‐LI secretion by luteal cells on days 11–13 and 14–18 of the cycle. Prolactin at doses of 0.1 and 1 ng/ml on days 11–13, and all doses tested on days 14–18 resulted in decreases in β‐END‐LI release from luteal cells. These results document evident changes in β‐END‐LI content in the pig corpus luteum during its development and indicate the potential roles of progesterone, oxytocin, and prolactin in luteal cell secretion of β‐END‐LI.     

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.     

Expression and Localization of Vascular Endothelial Growth Factor and its Receptors in the Corpus Luteum During Oestrous Cycle in Water Buffaloes (Bubalus bubalis)

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid‐luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non‐angiogenic function.     

Impact of Buserelin Acetate or hCG Administration on the Day of First Artificial Insemination on Subsequent Luteal Profile and Conception Rate in Murrah Buffalo (Bubalus bubalis)

This study was designed to investigate the impact of buserelin acetate (BA) or human chorionic gonadotropin (hCG) administration on the day of first artificial insemination (AI) on subsequent luteal profile (diameter of corpus luteum (CL) and plasma progesterone) and conception rate in Murrah buffalo. The present experiment was carried out at two locations in 117 buffalo that were oestrus‐synchronized using cloprostenol (500 μg) administered (i.m.) 11 days apart followed by AI during standing oestrus. Based on treatment (i.m.) at the time of AI, buffalo were randomly categorized (n = 39 in each group) into control (isotonic saline solution, 5 ml), dAI‐BA (buserelin acetate, 20 μg) and dAI‐hCG (hCG, 3000 IU) group. Out of these, 14 buffalo of each group were subjected to ovarian ultrasonography on the day of oestrus to monitor the preovulatory follicle and on days 5, 12, 16 and 21 post‐ovulation to monitor CL diameter. On the day of each sonography, jugular vein blood samples were collected for the estimation of progesterone concentrations. All the buffalo (n = 117) were confirmed for pregnancy on day 40 post‐ovulation. The conception rate was better (p < 0.05) in dAI‐BA (51.3%) and dAI‐hCG (66.7%) groups as compared to their control counterparts (30.8%). Furthermore, the buffalo of dAI‐hCG group had improved (p < 0.05) luteal profile, whereas the buffalo of dAI‐BA group failed (p > 0.05) to exhibit stimulatory impact of treatment on luteal profile when compared to control group. In brief, buserelin acetate or hCG treatment on the day of first AI leads to an increase in conception rate; however, an appreciable impact on post‐ovulation luteal profile was observed only in hCG‐treated Murrah buffalo.     

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.     

Short‐Term Undernutrition Affects Final Development of Ovulatory Follicles in Sheep Synchronized for Ovulation

The objective of this study was to determine, in sheep, the effect of a short‐term undernutrition on growth dynamics and competence of pre‐ovulatory follicles. Synchronization of sexual cycles and induction of ovulation were performed, with progestagens and gonadotrophins, in 14 adult female sheep. Morphological characteristics and developmental competence of ovarian follicles to achieve ovulation were determined by imaging techniques (ultrasonography and laparoscopy) and blood sampling. All the animals ovulated and mean ovulation rates were similar between groups (2.0 ± 0.6 corpora lutea in control ewes and 2.2 ± 0.8 in undernourished sheep). However, nutritional restriction, even during a short period, was related to the presence of large follicles in static growing phase which, despite reaching ovulation, persisted static during the induced follicular phase and evidenced functional alterations as there was no inhibition of the development of subordinate follicles. Thus, this study suggests the existence of deleterious effects from short‐term undernutrition on functionality of pre‐ovulatory follicles, which can compromise fertility.     

Enhancement of Ovulatory Follicle Development in Maiden Sheep by Short-term Supplementation with Steam-flaked Corn

The effect of a short‐term nutritional supplementation with steam‐flaked corn on metabolism and folliculogenesis was evaluated in 14 maiden sheep. Oestrus was synchronized with two prostaglandin F_2α doses given 10 days apart. From day 11 to 15 of the oestrous cycle induced with prostaglandins, half of the ewes (group 2M) were supplemented with steam‐flaked corn, double the daily maintenance ration of the control sheep (group 1M). Body weight and condition remained unaffected, but the energetic supply increased plasma concentrations of glucose (3.6 ± 0.1 vs 4.3 ± 0.1 mmol/l, p < 0.0001) for the first 4 days and 3‐hydroxybutyrate (0.323 ± 0.58 vs 0.582 ± 0.04 mmol/l, p < 0.005) from day 2 to 4. The profile of insulin secretion was also affected by the treatment, increasing in group 2M to reach significant differences on days 13 and 14 (p < 0.05). From similar values at the start of the food supply, the treatment induced a higher follicular development in group 2M (1.1 ± 1.2 vs 7.4 ± 1.06 total follicles in day 15, p < 0.05), as evidenced by the lineal increase in the number of larger follicles (>4 mm, p < 0.005). Then, the number of follicles >4 mm in size in 2M was around 60% higher on day 16 (7.86 ± 0.45 vs 4.86 ± 0.63, p < 0.005). Thereafter, the mean number of corpora lutea per ewe was around 30% higher in group 2M (1.43 ± 0.2 vs 1.10 ± 0.1, although differences were not found to be statistically significant). These data suggest that the use of diets containing high starch sources, like the steam‐flaked corn, increases folliculogenesis and ovulation rate in sheep and can be applied in short‐term feeding practices.