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This is the first time that PAG determination using two different antisera raised against PAG molecules purified from both caprine (RIA-706) and bubaline placentas (RIA-860) is reported in water buffalo. Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) programme, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and 45. Pregnancy was confirmed by ultrasonography on days 28 and 45. The blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 postcalving. Differences in PAG concentrations were observed between pregnant and nonpregnant buffaloes starting from day 23 post AI using both RIA-706 and RIA-860 (p < 0.001). However, estimated mean concentrations of PAG measured by RIA-706 were higher than RIA-860 (p < 0.001) and Bland-Altman analysis showed biases ranged from 0.0 ng/ml at day 23 to 0.79 ng/ml at day 28 post AI. Moreover, RIA-706 showed greater sensitivity and accuracy both at 23 and 25 days of pregnancy. RIA-706 and RIA-860 decreased below 1 ng/ml from 40 and 30 days postpartum, respectively, suggesting that PAG are better recognized by the antisera raised against the caprine PAG in the postpartum period also. This is essential when using PAG as an appropriate marker of early pregnancy after postpartum for detecting new pregnancies. The results of this study show that the ability of RIA systems to recognize early PAG could be improved using antisera raised against PAG molecules isolated from caprine placenta., (© 2018 Blackwell Verlag GmbH.)

For the first time in literature this study describes the pregnancy-associated glycoprotein (PAG) profile of buffalo cows during gestation and the post-partum period using antiserum raised against PAG-molecules purified from buffalo placenta (AS#860). Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and then biweekly until the end of pregnancy. Pregnancy was confirmed by ultrasonography on days 28 and 45, and by rectal palpation from day 60 onwards. Blood samples were suspended for the non-pregnant cows on day 45, while the blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 post-calving. A cut-off value of 1.0 ng/mL was used in order to discriminate between pregnant and non-pregnant buffaloes. We used Linear Mixed models after Log(x+1) transformation to analyse the PAG concentrations. Fifty-two buffalo cows had become pregnant out of 98 synchronized (53%) and 46 remained non-pregnant (47%) as shown by ultrasonography and the PAG analysis. Significant differences (P < 0.001) in PAG concentrations were observed between the pregnant and non-pregnant buffaloes from day 23 as the PAG of the non-pregnant cows was always close to zero. Conversely, the PAG of the pregnant cows increased progressively from day AI until day 105 post-insemination and then stabilized until the end of pregnancy. Regarding pregnancy diagnosis, the sensitivity of PAG-RIA 860 system (ability of the test to correctly identify pregnant buffalo) ranged from 23% on day 23-98% on day 28 post AI; the specificity (ability to correctly identify non-pregnant buffaloes) was 100% throughout the sampling period. PAG progressively decreased from parturition to day 25 post-partum; from day 30 post-partum, the concentrations fell below 1 ng/mL and were close to 0 on the last day of observation (50 d post-partum). In conclusion, our results showed that RIA-860 is highly accurate for diagnosing pregnancy in buffaloes starting from day 28 of gestation. Furthermore, the rapid disappearance of PAG concentration after calving means that a cut-off limit in post-partum for detecting a new pregnancy is not required., (Copyright © 2017. Published by Elsevier Inc.)

This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy in water buffalo. The PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer of the placenta in eutherian species. All PAGs are not concomitantly expressed throughout pregnancy; some of them are expressed in the earlier phases, whereas others appear later and are expressed over a shorter period. Twenty-one lactating buffaloes were analyzed-17 females were synchronized with PRID and artificially inseminated (AI), whereas four females were synchronized but not inseminated (control group). Blood was collected at Days 0, 18, 28, 40, and 75 from AI (AI = Day 0). Expression of PAG-2 mRNA in blood samples was measured with real-time polymerase chain reaction. Pregnancy diagnosis was performed on Day 28 (D28) and Day 40 (D40) after AI by ultrasonography (US) and by PAG-1 RIA method. The females diagnosed pregnant at D28 and confirmed pregnant at D40 were defined as D28(+)D40(+) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(+)D40(-) group; and the females that were diagnosed as nonpregnant on either days were defined as D28(-)D40(-) group. PAG-2 mRNA at Day 0 was not observed in any groups. The D28(+)D40(+) group showed the highest expression, starting on Day 18 and increasing progressively up to Day 75. PAG-2 mRNA was also expressed on Day 18 in both D28(+)D40(-) and D28(-)D40(-) groups, but their levels were lower than those of D28(+)D40(+) group and almost constant over time. PAG-2 mRNA was never detected in the control group. The significant difference in the expression of PAG-2 mRNA between the D28(+)D40(+) group and the D28(-)D40(-) group, starting from Day 18, suggests that these animals might have conceived, but have experienced early embryonic loss; therefore, the PAG-2 mRNA was still present in blood circulation although at lower levels, as found in the D28(+)D40(-) group. In conclusion, this study shows that PAG-2 mRNA can be detected in peripheral maternal blood cells earlier than circulating PAG-1 molecules and could be useful for studies on early pregnancy and embryonic mortality., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to describe modifications in plasma homocysteine (Hcy), vitamin E (VitE) and malondialdehyde (MDA) concentrations in the first 56 days after artificial insemination (AI) in buffalo. Thirty-five buffalo cows were divided, ex post, into three groups on the basis of pregnancy diagnosis: pregnant, not pregnant, with embryonic mortality. Pregnancy was diagnosed by ultrasonography and plasma concentrations of pregnancy-associated glycoproteins (PAGs). Our results showed that, in pregnant buffaloes, included those with embryonic mortality, MDA increased progressively while VitE decreased. In non-pregnant buffaloes, MDA and Vit E were unchanged. Hcy concentrations also remained unchanged within each group throughout the study period, but were lower in non-pregnant buffaloes than in the pregnant ones and in those with embryonic mortality. In conclusion, present data suggest that successful pregnancy in buffalo cows might be linked to Hcy metabolism and oxidative stress involvement., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine the timing of ovulation in relation to the LH peak after synchronization using PRID or Ovsynch protocols, to assess the effects of the period of treatment on these parameters and to provide information concerning how to use the two main protocols for fixed-time artificial insemination in buffalo. Forty-eight lactating Italian Mediterranean buffalo cows were used. The buffaloes were treated in various periods as follows: February to March (n = 12 PRID, n = 12 Ovsynch), end of the breeding season, May to June (n = 12 PRID, n = 12 Ovsynch), beginning of low-breeding season according to Italian environmental conditions. To determine the LH, blood samples were taken at 4-hour intervals, starting 24 hours from PRID removal (PRID group) or 12 hours from (PGF2α) injection (Ovsynch group) up to 108 hours. The ovaries were monitored by transrectal ultrasonography to verify ovulation. The LH-ovulation interval was similar in both groups (30.10 ± 1.05 and 32.77 ± 1.15 hours, respectively, in PRID and Ovsynch group). In the PRID group, the timing of ovulation in relation to device removal was 76.83 ± 3.65 hours with a high level of variability among the animals. In the Ovsynch group, we observed a better synchronization of LH peaks and ovulations, and the timing of ovulation in relation to the last GnRH injection was 35.67 ± 1.15 hours. The percentage of animals reaching the LH peak and ovulation was lower (P ≤ 0.05) in May to June (respectively 75.0% and 54.1%) compared to February to March (respectively 95.8% and 83.3%), indicating a reduction of hypothalamus-pituitary responsiveness to the synchronization treatments in the daylight-lengthening period., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In order to evaluate the influence of rearing system (free-ranging (FR) v. confinement (C)) on buffalo heifer efficiency to reach age of puberty and on behavioural and immune functions, two experiments were conducted from September 2010 to October 2011. In Experiment I, 32 subjects aged 8 to 9 months at the start of experiment were used. A total of 16 animals (group C) were group housed in an indoor slatted floor pen (4 m2/animal) with an outdoor paddock (4 m2/animal); 16 others grazed on a Mediterranean natural pasture of 40 ha (group FR). Behavioural data were collected and organic matter digestibility, blood metabolites and progesterone were determined. At the end of the experiment, a novel object test and a skin test were conducted, and the avoidance distance (AD) at the manger was measured. Free-ranging animals were able to express natural behaviours such as wallowing and grazing. C animals devoted more time to the novel object than FR animals, whereas AD at manger was lower in group FR than in group C (P<0.01). Cellular immune response was higher in FR heifers than in C animals (P<0.01). FR animals also showed a higher digestibility of organic matter (P<0.01). Heifers from group FR had higher plasma concentrations of non-esterified fatty acids (P<0.001) and lower concentrations of glucose than heifers from group C (P<0.001). C animals showed higher daily weight gains (P<0.01) and weight at the puberty (P<0.05), but there were no differences in terms of age of puberty between the two groups. The intakes of dry matter (DM), CP and energy to reach the age of puberty were similar in both groups. In order to verify whether the results obtained in Experiment I could be replicated in different rearing conditions (reduced pasture availability, different location and altitude), a second experiment was conducted on 26 animals, where only onset of age of puberty and metabolic profile were monitored. In Experiment II, 13 heifers grazed on a natural pasture of 5 ha, other 13 received the same space as indicated for Experiment I. Results from Experiment II generally confirmed those of Experiment I. Only the intakes of DM and energy to reach the age of puberty were higher in group C than in FR (P<0.001). A lower competition with human nutrition, reproductive performances similar to those shown by confined animals and the indications given by immune and behavioural variables, suggest that a free-range-based system may be conveniently used for buffalo heifer farming purposes.

Background: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species., Results: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n=33) and non-pregnant (n=26) water buffalo females., Conclusions: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen-thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR-14/propidium iodide staining; mitochondrial function by JC-1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC-PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψ(high)), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %-DFI, -DFI, SD-DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome-reacted live (ARL) and acrosome-reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca(2+) ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential., (© 2012 Blackwell Verlag GmbH.)

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

The primary objective was to elucidate ovarian follicular dynamics and hormonal profiles in nulliparous heifer (HE; n = 11 ) and mixed-parity (MP; n=10 ) Mediterranean Italian water buffaloes (Bubalus bubalis) following an estrus synchronization protocol. Both groups received a progesterone releasing intravaginal device (PRID) implant for 10 days; a luteolytic dose of synthetic prostaglandin was given 7 days after PRID insertion. Daily ultrasound monitoring and collection of blood to determine plasma concentrations estradiol and progesterone started 1 day after PRID removal and lasted for 55 and 65 days in HE and MP buffaloes, respectively. Data analysis was restricted to the first 5 days after PRID removal and to one estrus cycle following induced ovulation. The HE buffaloes were not inseminated and only one ovulated within 5 days after PRID removal; the remainder ovulated between 8 and 48 days after PRID removal (except one in which ovulation was never detected). All HP buffaloes were inseminated 72, 96 and 120 h after PRID removal; seven buffaloes ovulated within 5 days after PRID removal and two were pregnant. Mean diameter of the largest follicle was significantly smaller in HE than MP buffaloes the first 4 days after PRID removal. There was a parity by time interaction ( P=0.0047 ) for plasma progesterone concentrations; progesterone was higher in HE than MP buffaloes 1 day after PRID removal, but the converse was true 2 days after PRID removal. After induced ovulation, HE buffaloes exhibited a one-wave ( n=5; length of cycle, 8-12 days), two-wave ( n=4; range: 20-26 days) or three-wave cycle ( n=1; 25 days). In contrast, all non-pregnant MP buffaloes ( n=8 ) had a two-wave cycle (range: 19-25 days). For buffaloes with two-wave cycles, the growth rate and diameter of the largest follicle was significantly smaller in HE than MP buffaloes for both the first follicular wave (1.3mm versus 1.7 mm per day and 10.5 mm versus 13.3 mm, respectively) and the second follicular wave (1.0 mm versus 1.3 mm per day and 11.0 mm versus 13.8 mm). In conclusion, there were many significant morphological and endocrine differences between HE and MP buffaloes.

The aim of this study was the investigation of hormonal and ovarian follicular dynamics in prepuberal buffaloes (Bubalus bubalis) bred in Italy. Eleven 5-9-month old buffalo calves ranging in weight from 122 to 270kg, maintained under controlled nutritional and environmental conditions, underwent 50 days of ultrasonographic ovarian follicular monitoring in the months of October-December. Blood sampling for E(2) and FSH determination and ultrasonographic monitoring using a 7.5MHz linear probe and an ALOKA SSD-500 monitor were performed daily. No differences in any of the parameters under study were highlighted when calves were divided into two weight categories (<200 and >200kg) and thus data were pooled. In this study, values are reported as mean+/-S.D. A range of two-six regular follicular waves was reported among calves with an average of 4+/-1.1. Overall interval (days) between wave emergence was 9.9+/-2.8 and largest diameters (mm) of dominant and first subordinate follicles were 8.4+/-1.2 and 4.8+/-0.6, respectively (P<0.05). With the exception of one calf, some minor follicular waves (short waves or SWs; 1.6+/-1), lasting <10 days (6.1+/-1.2) were reported. They were monitored contemporaneously on the ovary contralateral (n=7) or ipsilateral (n=3) to the main follicular wave. Growth rate (mm per day) of dominant follicles (DF) was significantly faster than for corresponding subordinate follicles (SF) and follicles of SWs (1.08+/-0.2 versus 0.79+/-0.1 and 0.83+/-0.1, respectively, P<0.05). The static phase (days) lasted longer in DF compared to SF and SW (5.4+/-1.8 versus 2.4+/-1.2 and 2.6+/-1, respectively, P<0.05). The regressing phase (mm per day) was similar among DF, SF and SW (0.86+/-0.2, 0.94+/-0.2 and 0.84+/-0.1, respectively, P=0.09). Episodic spikes of E(2) and FSH were reported, corresponding to wave development throughout the course of investigation. In conclusion, the majority of buffalo calves displayed a typical pattern of regular follicular development in conjunction with a dynamic trend of ovarian and hypophyseal hormones. Some minor follicle turnover was reported with parallel main follicular waves.