Teat morphology across five buffalo breeds: a multi-country collaborative study.

The buffalo (Bubalus bubalis) is a species of worldwide importance, raised to produce milk, meat, and hides, and often used as a working animal in rural contexts with low access to hi-tech solutions. In the present study, 100 lactating buffaloes (50 primiparous and 50 pluriparous) of five popular breeds were recruited to characterize and compare teat morphology. In particular, the focus was put on the Nili Ravi, Mediterranean, Egyptian, Bulgarian Murrah, and Azeri buffaloes raised in Pakistan, Italy, Egypt, Bulgaria, and Iran, respectively. In all countries, a longitudinal cross-section ultrasound was obtained before the milking to measure teat parameters at individual level: overall, teat canal length (TCL) averaged 24.13 mm, teat diameter (TD) 30.46 mm, cisternal diameter (CD) 17.80 mm, and teat wall (TW) 7.12 mm. The most variable trait across breeds was TCL which was positively correlated with CD and TD and negatively with TW, regardless of the teat position (front/rear or left/right). A strong negative correlation was found between TW and CD (- 0.43). The analysis of variance revealed that the fixed effect of breed significantly affected all the traits except TD. In fact, Bulgarian Murrah, Azeri, and Egyptian buffaloes presented the greatest estimate of TCL, whereas NR the smallest (14.70 mm). The TW was maximum in Nili Ravi, Egyptian, and Mediterranean buffaloes, with estimates equal to 8.19, 7.59, and 8.74 mm, respectively. Nili Ravi also showed the greatest TL (82.39 mm). In terms of CD, the lowest least square mean was that of Mediterranean buffaloes (12.14 mm). Primiparous and pluriparous buffaloes differed in terms of TD, TW, and TL, with older animals presenting the highest least square mean. In terms of position, instead, significant differences were observed for TD, CD, and TL when comparing front and rear teats, as left and right teats did not differ. Teat anatomy includes a set of heritable morphological features and is therefore breed-dependent. Differences presented in this study could be attributed to the divergent breeding objective and selective pressure across the five breeds; e.g., in some cases such as Mediterranean buffalo, selection for decades was oriented to improve milk production and milkability and achieve optimal conformation for mechanical milking. A better understanding of the mammary gland anatomical descriptors can be informative of the history of a breed and could provide useful insights to guide possible selection.

Effects of extender type and storage time on sperm quality parameters of Kail ram semen stored at 5 °C.

The study was designed to evaluate the effect of different extenders and storage times on sperm quality parameters of extended Kail ram semen. Semen was collected from five adult Kail rams using an artificial vagina. Semen samples with >70% total sperm motility were pooled, diluted with Tris (TR), sodium citrate (SC), and skim milk (SM)-based extenders, and stored at 5 °C. Sperm motility and kinematics, viability, and plasma and acrosomal membrane integrity were assessed every 24 hrs for 120 hrs. Sperm longevity was estimated by placing semen in a water bath at 37°C to assess sperm kinematics. Storage time as the main effect had a negative (P < 0.05) impact on sperm quality parameters. The percentages of total motile (TM), progressive motile (PM), rapid, and medium progressive (MP) motile sperm were similar at 24 hrs of storage for the three extenders. However, semen extended in TR had a higher (P < 0.05) percentage of TM, PM, rapid, and MP sperm compared to SM and SC extenders at 48 hrs of storage. The sperm kinematics (VCL, VSL, VAP, and ALH) of progressive sperm were similar for all three extenders up to 72 hrs of storage. In addition, semen extended in TR had a higher (P < 0.05) percentage of sperm with intact plasma and acrosomal membranes at a storage time of 48 hrs. At 37 °C, the percentage of TM and PM sperm was higher (P < 0.05) in the TR extender than in the SM and SC extenders at 60 minutes and beyond. In conclusion, the Kail ram sperm extended in TR and stored at 5 °C maintained better quality after 48 hrs storage than sperm extended in SM and SC extenders. At 37 °C, sperm extended in TR also retained better quality parameters at 60 min and afterward than sperm extended in SM and SC.

Exogenous progesterone-dependent modulation in the follicular dynamics of Bos indicus cattle undergoing repeated ovum pick-up sessions.

The objective of the present study was to evaluate the role of serum progesterone (P4) in follicular dynamics, oocytes' recovery and quality and their in vitro developmental competence during consecutive ovum pick-up (OPU) sessions in Bos indicus dairy cows. Wave-synchronized Sahiwal cattle (n = 20) were randomly divided into treatment (n = 10) and control (n = 10) groups. CIDR was used as a source of external progesterone in the treatment group. Four consecutive OPU sessions at 96-hr intervals were conducted and repeated ultrasonography at 12-hr intervals was done to monitor follicular dynamics. The viable oocytes were processed for IVC following IVM and IVF until day 7. The serum P4 concentrations in the P4 and control groups were recorded as 2.31 ± 0.059 versus.0.32 ± 0.065 ng/ml, respectively (p < .05). In the treatment group, the total number of recorded follicles was higher (p < .05; 12.05 ± 0.37 versus. 10.87 ± 0.40), whilst the growth rate (mm/day) of follicles was lower (p > .05). Per session recovered oocytes (5.31 ± 0.19 versus. 3.58 ± 0.21; p < .0001) and recovery rate (54.23 versus. 42.53%; p < .05) were also higher in the treatment group compared to control. Similarly, the viable oocytes (4.54 ± 0.187 versus. 3.06 ± 0.199) and the number of grade I and II oocytes per session (3.37 ± 0.196 versus. 2.06 ± 0.21) were higher (p < .05) in the treatment group compared with the control group. However, the nuclear maturation, cleavage, and blastocyst rate did not differ (p > .05) between the groups. Taken together, during OPU sessions, serum P4 improves oocytes' recovery and quality, whilst does not affect the in vitro developmental competence of recovered oocytes.

Effect of exogenous administration of oxytocin on postpartum follicular dynamics, oestrous rate and ovulation in Nili-Ravi buffaloes.

Based on different surveys, dairy farmers are concerned about extensive use of exogenous oxytocin in buffaloes, which is being held responsible for reproductive problems including irregular oestrous cycle and delayed ovulation. For these concerns, effects of oxytocin injection on postpartum follicular dynamics, postpartum oestrous interval (PEI), oestrous length, the interval from onset of estrus to ovulation and blood progesterone (P4) were studied in Nili-Ravi buffaloes. For this purpose, 23 animals within 1 week after calving were randomly divided into three groups: without oxytocin (CON; n = 7), 10 i.u. oxytocin (LOW; n = 8), 30 i.u. oxytocin - (HIGH; n = 8) and used to record the PEI for the study period of 154 days. At subsequent estrus, three buffaloes from each group (not served) were selected randomly to monitor two cycles for 6 weeks. Transrectal ultrasonography was performed to evaluate follicular and corpus luteum (CL) development, and blood sampling was done for progesterone (P4) analysis. These results revealed that postpartum oestrous interval (PEI) decreased significantly in oxytocin-treated groups. The number of small, medium and total follicles on the left ovary was significantly higher in the HIGH group. However, an overall number of small and total follicles on both right and left ovaries was significantly higher in CON and HIGH groups. On the other hand, there was no difference in the number of follicles on the right ovary among all treatment groups. The same was true for the size of pre-ovulatory follicles, CL, P4 concentrations and oestrous cycle length. The intervals from onset of estrus to ovulation and from standing estrus to ovulation were increased considerably in the HIGH group. It is concluded that exogenous oxytocin administration resulted in the shortening of PEI but triggered a delay in ovulation. Moreover, a higher dose of oxytocin could stimulate the growth of small, medium, and total follicles in postpartum Nili-Ravi buffaloes.

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.

Luteal blood flow measured by Doppler ultrasonography during the first three weeks after artificial insemination in pregnant and non-pregnant Bos indicus dairy cows.

The objective of this study was to determine if there are differences in luteal size (LS), progesterone (P4), and luteal blood flow (LBF) between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after insemination, and whether these parameters are related to each other. Lactating cows (n = 13) of mixed parity with a body weight of 430 ± 18 kg (mean ± SD), showing regular estrous cycle were used in the study. All cows were artificially inseminated and were classified as pregnant (embryonic heartbeat on day 30; n = 8) or non-pregnant (inter-estrus interval 17 to 21 days, n = 5). In order to compare the LS and LBF after artificial insemination, B-mode and color Doppler ultrasonography of ovaries were performed on days 4, 5, 6, 7 (first week), 8, 10, 12, 14, (second week), and 16, 17, 18, 19, 20, 21 (third week) in pregnant and non-pregnant cows. Results revealed that the mean LBF was consistently higher (P < 0.05) during days 7 through 21 in pregnant cows than in non-pregnant cows. The mean LS was higher (P < 0.05) on days 6 and 7, and from day 17 onwards, and the mean concentration of P4 was higher (P < 0.05) on days 19, 20, and 21 in pregnant cows. In conclusion, LBF is a more sensitive parameter than LS and P4 for detection of differences in luteal function between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after AI.

Effect of breeding method and season on pregnancy rate and embryonic and fetal losses in lactating Nili-Ravi buffaloes.

The aim of this study was to determine the effect of breeding method and season on pregnancy rate and cumulative embryonic and fetal losses in Nili-Ravi buffalo. Estrus detection was performed twice a day by teaser buffalo bull for 1 hour each. A 2 × 2 factorial design was used to address the breeding method and season. Buffaloes (n = 130) exhibiting estrus were randomly assigned to be bred either in peak breeding season (PBS; n = 80) or low breeding season (LBS; n = 50). Within each season, buffaloes were divided to receive either natural service (NS; n = 65) or artificial insemination (AI; n = 65). NS buffaloes, in estrus, were allowed to remain with the bull until mating. AI was achieved, using frozen thawed semen of bull of known fertility. PBS comprised of September to December and LBS were from May to July. Serial ultrasonography was performed on days 30, 45, 60, and 90 after breeding (day 0) to monitor pregnancy rate and embryonic and fetal losses. The pregnancy rate on day 30 after breeding was higher in NS as compared to AI group (63 vs. 43%; P < 0.05) during PBS while it did not differ (48 vs. 32%; P > 0.05) in LBS. The cumulative embryonic and fetal losses between days 31 and 90 were significantly lower in PBS than LBS (33 vs. 60%; P < 0.05), ignoring breeding method. Pregnancy rates were better with NS in PBS, and cumulative embryonic fetal losses were higher in LBS in Nili-Ravi buffalo.

Incorporation of estradiol benzoate to CIDR protocol improves the reproductive responses in crossbred dairy heifers.

The present study was designed to determine the effect of estradiol benzoate (EB) on reproductive response following a controlled internal drug release (CIDR) protocol in crossbred (Sahiwal × Friesian) dairy heifers. In the first trial, a total of 100 crossbred dairy heifers were treated with CIDR protocol for 7 days and injected with the PGF2α on day 6. After 24 h of CIDR removal, one group (EB = 50) was injected with estradiol benzoate whereas the other (control = 50) remained untreated. Estrus intensity and response were recorded visually and ovulation rate was recorded by ultrasonography. All heifers were artificially inseminated at 48 and 60 h following CIDR removal. Heifers were scanned for pregnancy within days 30-40 of artificial insemination (AI). In the second trial, two subgroups of heifers were included to observe the estrus and ovulatory events. The results of the first trial revealed that estrus response was achieved 100% in both the treatment groups. Estrus intensity (2.9 ± 0.1 vs. 2.0 ± 0.7) and ovulation rate (100 vs. 88%) differed significantly (P < 0.05) between the EB and control groups. However, a tendency for higher pregnancy per AI was observed (54 vs. 36%; P = 0.07) in EB than that in control groups. The results of the second trial revealed that a significantly (P < 0.05) shorter estrus and earlier ovulatory events were observed in EB-treated heifers. It is concluded that the incorporation of estradiol benzoate to the CIDR protocol is helpful to improve the estrus signs and enhance the ovulation and the pregnancy per AI in crossbred dairy heifers.

Sperm survival kinetics in different types of bull semen: progressive motility, plasma membrane integrity, acrosomal status and reactive oxygen species generation.

This study was designed to compare the kinetics of sperm survival in different types of bull semen. Fresh ejaculates from four bulls were pooled, diluted in Tris-citric acid-egg yolk-glycerol extender, cooled to 4°C, frozen in LN2 and thawed at 37°C. Fresh, diluted, cooled and frozen-thawed semen were incubated at 37°C, and evaluated at 0, 2, 4, 6, 12 and 24h after the beginning of incubation. In Experiment 1, progressive sperm motility, normal acrosomes and plasma membrane integrity and asymmetry were determined. In Experiment 2, generation of superoxide anion (O2(•)) along with plasma membrane permeability and generation of hydrogen peroxide (H2O2) along with plasma membrane integrity were assessed. In Experiment 1, frozen-thawed semen had shorter survival times for progressive sperm motility, and spermatozoa with intact plasma membranes and acrosomes (IPM-IACR) as compared with other types of semen (P<0.05). Fresh spermatozoa underwent a necrotic pathway, diluted and cooled spermatozoa underwent an apoptosis-like pathway and frozen-thawed spermatozoa underwent both necrotic and apoptosis-like pathways. In Experiment 2, spermatozoa in all four types of semen exhibited O2(•-) generation and increased plasma membrane permeability, and became necrotic without H2O2 generation during incubation (P<0.05). In conclusion, frozen-thawed semen had shorter sperm longevity, which has important implications relating to the timing of artificial insemination. Different types of semen followed different death pathways. During incubation, spermatozoa in all types of semen generated O2(•-), which increased the permeability and compromised the integrity of the plasma membrane.

Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm.

Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8h) on survival before freezing at 4°C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates=6), pooled and diluted with Tris-citrate extender. Pooled semen samples were subjected to either RC (-2.2°C/min) or MC (-0.3°C/min) from 37°C to 4°C in separate aliquots and further equilibrated at 4°C for 8h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4°C, of motility and PMI was observed 5 and 6h respectively in RC group while >8h in MC group. Rate of decline (slope) in motility and viability was higher (P<0.05) in RC overtime during equilibration at 4°C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P<0.05) in MC when equilibrated for 2-8h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2-8h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.

Effect of Milk Type Subjected to Different Heat Treatments on Cryo-Survivability and In Vivo Fertility of Buffalo (Bubalus bubalis) Spermatozoa in a Milk-Based Extender.

Oxidative stress is a major contributory factor to cellular damage during semen cryopreservation and results in a decreased fertilizing capacity of cryopreserved bull sperm. The inclusion of exogenous antioxidants sometimes exerts deleterious effects on sperm quality. Thus, enhancing the endogenous production of antioxidants is a requirement. This study aimed to investigate the effect of milk type heated at different temperatures on the antioxidant potential of extenders, and the subsequent post-thaw quality parameters and in vivo fertility of buffalo bull semen. Cow (C) and buffalo whole milk (B) were used separately for semen extender preparation, heated at five different temperatures (T1 = 90°C, T2 = 100°C, T3 = 110°C, T4 = 120°C, T5 = 130°C) for 10 minutes. Reactive sulfhydryl groups were measured in each subgroup by Ellman's reagents as CT1 = 143.2 µM, CT2 = 147.4 µM, CT3 = 151.5 µM, CT4 = 157.2 µM, CT5 = 161.8 µM, BT1 = 168.3 µM, BT2 = 172.5 µM, BT3 = 176.7 µM, BT4 = 196.3 µM, and BT5 = 205.7 µM. All semen samples were cryopreserved in milk-based extenders by using standard procedures. Post-thaw quality parameters including total and progressive motility, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity were found to be higher (p < 0.05) in the group (BT3) containing buffalo milk heated at 110°C, whereas in the same group, lipid peroxidation was found to be lower (p < 0.05) as compared with other treatment groups and control group. In vivo fertility of cryopreserved buffalo sperm was compared among BT3, CT1 (conventionally used milk extender), and a Tris egg yolk extender group. The fertility rates [47% (54/114), 30% (33/108), and 36% (37/103)] were higher (p < 0.05) in BT3 as compared with other groups. This study suggests that buffalo milk heated at 110°C has high antioxidant potential and improves post-thaw quality and in vivo fertility of cryopreserved buffalo bull semen.

Broad-spectrum resistance against multiple PVY-strains by CRSIPR/Cas13 system in Solanum tuberosum crop.

Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.

Relationship of age to body weight, scrotal circumference, testicular ultrasonograms, and semen quality in Sahiwal bulls.

The objective of the present study was to determine the relationship of age to body weight (BW), scrotal circumference (SC), number of pixels of testicular ultrasonograms (NP), and semen quality in Sahiwal bulls. The study was based on 128 Sahiwal bulls of different age groups (from 0 to >100 months of age). Bulls were evaluated for SC, BW, and NP. Semen was evaluated once a week for five consecutive weeks from regularly collected donor bulls (n = 86) ranging in age from 25-30 to >100 months. Ejaculate volume, sperm motility, sperm concentration, sperm morphology, percent live sperms, sperm plasma membrane integrity, and normal acrosome were compared among different age groups. Mean SC and BW increased (P < 0.05) in a curvilinear manner from birth to >100 months of age. Mean NP of testicular ultrasonograms increased (P < 0.05) from 0 to 24 months and then plateaued until >100 months of age. Body weight, SC, and NP were positively correlated with age from birth until >100 months (r = 0.91, 0.87, and 0.40, respectively). Ejaculate volume (5.7 ± 0.2 vs. 4.6 ± 0.09 ml) and sperm concentration (1,281.6 ± 17.7 vs. 1,115.8 ± 55.9 × 10(6)/ml) increased (P < 0.05) in mature bulls compared to younger ones. However, motility (68.6 ± 0.3%), plasma membrane integrity (50.8 ± 1.0%), and normal acrosome (74.8 ± 0.5%) remained insignificant due to age. In six of eight age groups studied, morphological abnormalities were well within the range (18.1 ± 0.3%). In conclusion, the BW, SC, and NP of testicular ultrasonograms, ejaculate volume, and concentration increased with age. Moreover, semen quality is fairly independent of age except volume and concentration in Sahiwal bulls.

Genotyping of Brucella strains isolated from humans and cattle of different geographical regions of Pakistan using MLVA-15.

BACKGROUND: Prevalence of brucellosis and MLVA genotyping in animals and humans, isolated from different regions of Pakistan was performed. Animals having history of brucellosis from the field and local farms of Bannu, Mardan, Peshawar, Swat, Lahore and Islamabad were selected for blood collection. Humans that work with them were also selected for sampling in this study. Total of 600 samples were taken from cattle and humans and subjected to Rose Bengal plate Test (RBPT) for the initial screening of positive samples. Designed primers of B.abortus for cattle and B.melitensis for humans were utilised to perform PCR. Culturing and isolation was carried to further to perform MLVA genotyping assay through the selection of two panels of primer markers. RESULTS: RBPT showed more number of cases of brucellosis in animals and humans compared to the PCR findings. Genotyping findings based upon MLVA-15 set of markers demonstrated that the isolated strains of B.abortus fall in the same clade with strain A1, P8 and A2 from Pakistan and also similar with BCCN#02-45 strain from India. On the other hand, B.melitensis isolated from different districts of Pakistan shared the same clade with BwIM-AFG 63, BwIM-AFG 44 strains from Afghanistan and BwIM IRN 37 strain from Iran. Selected VNTR alleles were sequenced for calibration purposes. CONCLUSION: It is concluded that Brucella is prevalent in animals and humans in studied districts of Pakistan. Moreover, A1, P8, BwIM-AFG 63, BwIM-AFG 44 and A2 were found the common genotypes in Pakistan.

Synchronization and resynchronization strategies to improve fertility in dairy buffaloes.

Dairy buffalo has an integral role in the sustenance of economics due to its substantial contribution in milk and meat industry, however, the reproduction in this species is challenging. During the last decade, our laboratory conducted a series of experiments to encapsulate the solutions of the problems through optimizing pre- and post-insemination interventions in dairy buffaloes. In an unique study, we proposed that timing of ovulation with reference to the onset of standing heat during spontaneous estrus is delayed, and subsequently re-framed the traditional AM-PM rule (AI after 12 h of standing heat) to AM-AM or PM-PM (AI after 24 h of standing heat) to achieve the optimum fertility using frozen thawed semen in dairy buffaloes. Pregnancy per AI (P/AI) varied substantially either via injecting single shot of prostaglandin (PG) F2α to perform AI at detected estrus or applying standard ovsynch protocol for timed AI (TAI) in buffaloes. However, estrus response, and P/AI remained similar either with used or new controlled internal drug release device in dairy buffaloes. Additionally, the incorporation of estradiol benzoate in progesterone (P4) based protocol resulted in controlled emergence of follicular wave and increased the estrus intensity in buffaloes. Thereafter, we fine-tuned P4-based protocols to optimize the ovulation window for TAI either using GnRH or human chorionic gonadotropin (hCG) or equine chorionic gonadotropin that ultimately improved the fertility in dairy buffaloes. Although, these hormonal interventions resulted in decent fertility, yet it was consistently being compromised due to early or late embryonic losses in dairy buffaloes. Administration of hCG or GnRH on d 7 or 23 or 25 post AI has been proved beneficial to enhance the embryonic survival in buffaloes. Recently, resynchronization program as an aggressive reproductive management approach has been tested that served as a dual-purpose tool to increase overall herd fertility and reduce embryonic losses at commercial buffalo farm operations. Taken together, we concluded that the solutions to the problems of reproductive function are now clearly available with acceptable fertility, however, their application to the small holder buffalo farming remains challenging.

Effect of follicle size on oocytes recovery rate, quality, and in-vitro developmental competence in Bos indicus cows.

The objective of the present study was to determine the effect of follicle size on recovery rate, quality, and in-vitro developmental competence of oocytes in Bos indicus cows. The ovaries (n = 507) of Bos indicus cows having age of 5-8 years, with mixed parity, BCS 2.75 ± 0.25, and clinically normal reproductive tracts were collected from the local abattoir. The follicles on the ovaries were divided into two groups based upon their size; 1) ≥6 mm diameter, and 2) <6 mm diameter. After initial evaluation of quality of the oocytes, the COCs were in vitro matured, fertilized, and cultured to determine the in vitro developmental competence. The oocyte recovery, quality, maturation, cleavage, 4-cell, 8-cell, and 16-cell stages were analyzed using PROC GLIMMIX procedure of SAS. However, the number of oocytes recovered per ovary was analyzed using MIXED procedure of SAS. Results revealed that the recovery of oocytes (LSM ± SEM) derived from the follicles having size <6 mm per ovary was greater (1.02 vs. 3.14 ± 0.13; P < 0. 0001). However, the percentage (n/n) recovery [69.8 (474/679) vs. 62.7% (1454/2320); P = 0.01] and grade I_+_II oocytes [68.4 (324/474) vs. 57.9% (842/1454); P < 0.0001] was greater in ≥6 mm as compared with <6 mm group, respectively. However, maturation rate did not differ [92.9 (288/310) vs. 92.2% (296/321); P = 0.98] between the groups. In contrast, cleavage rate [58.1 (180/310) vs. 47.4% (152/321); P = 0.01], the 4-cell [34.5 (107/310) vs. 18.7% (60/321); P = 0.0003], 8-cell [15.5 (48/310) vs. 7.8% (25/321); P = 0.008], and 16-cell [8.7 (27/310) vs. 2.1% (7/321); P = 0.004] stage embryos were greater in ≥6 mm group. It can be concluded that oocytes derived from follicle ≥6 mm have better in vitro developmental competence based on embryonic conversion in Bos indicus cows.

Effect of photoperiodicity and methods of follicular wave emergence on follicle turn-over, recovery and quality of oocytes, and early in-vitro developmental competence of embryos using ovum pick-up in Nili-Ravi buffaloes: Preliminary evidence.

The objectives of the present study were to evaluate the effects of photoperiodicity, gauge (G) of ovum pick-up (OPU) needle, and two methods of follicular wave emergence on follicular turn-over, oocyte recovery (OR), quality of the oocytes (OQ), and early in-vitro developmental competence of embryos in Nili-Ravi buffaloes (n = 20). In 1st experiment, buffaloes (n = 12; 4 buffaloes/season) were randomly assigned to optimize the OPU's (n) either with 17 G or 18 G needle in one of the following seasons: 1) peak breeding season (PBS; Sep-Nov; n = 31), 2) transition breeding season (TBS; Dec-Feb; n = 32), and 3) low breeding season (LBS; Apr-June; n = 32). During 2nd experiment, buffaloes (n = 8) were enrolled randomly in a 2 × 2 cross-over design to compare the two methods of wave emergence either using follicular ablation (FA; n = 4), or synchronization protocol (CIDR-EB; n = 4) during PBS. In FA method, the ovarian follicles were aspirated (week -1), and subsequently repeated OPU's (n = 55) were performed for 7 weeks. However, in CIDR-EB synchronized buffaloes, a progesterone device (CIDR) was inserted in the anterior vagina and a single dose of estradiol benzoate (2 mg) and prostaglandin (150 µg) were administered i.m. on d 1. The CIDR was removed on d 7 and repeated OPU's (n = 56) were performed. In both experiments, a 7-d resting period was provided between each OPU session. Data on the follicular turn-over, OR, OQ, and early stages of embryonic development were analyzed with mixed models using the PROC MIXED and GLIMMIX procedures of SAS. Results revealed that the number of medium sized follicles (LSM ± SEM) and rate of OR (%) were greater (P < 0.05) during PBS (2.76 ± 0.40; 61%) and TBS (1.73 ± 0.30; 54%) as compared to LBS (0.68 ± 0.30; 31%) in buffaloes, respectively. The OR rate was also greater (P < 0.05) using 17 G as opposed to 18 G needle (62% vs. 35%), however, there was no effect (P > 0.05) of season and G of needle on OQ in buffaloes. In experiment 2, the number of small (3.50 ± 0.63 vs.2.69 ± 0.60) sized follicles was higher (P < 0.05) in FA, whereas, medium (1.13 ± 0.45 vs. 1.59 ± 0.45) sized follicles were greater (P < 0.05) in CIDR-EB synchronized buffaloes. The OR rate (67% vs. 53%), and OQ (Grade I_+_II; 15% vs. 16% and Grade III_+_IV; 85% vs. 84%), remained similar (P > 0.05) in FA and CIDR-EB treated buffaloes, respectively. Similarly, rates of in-vitro maturation (66% vs. 59%), cleavage (42% vs. 53%), 4-cell (27% vs. 32%), 8-cell staged embryos (23% vs. 25%), and morula (13% vs. 8%) did not differ (P > 0.05) between FA and CIDR-EB methods of follicular wave emergence, respectively. Taken together, it is concluded that peak or transition breeding seasons are suitable to perform OPU using 17 G needle for maximum follicular turn-over, and OR in buffaloes. However, the two fundamental methods of synchronization of wave emergence resulted in similar efficiency for OPU derived in-vitro embryo production in Nili-Ravi buffaloes.

An Observational Registry to Assess Urinary Albumin Evolution in Saudi Hypertensive Patients with the Current Treatment Local algorithm: Results of the RATIONAL Study.

INTRODUCTION: Hypertension causes microalbuminuria, which if left uncontrolled could progress to kidney damage. Antihypertensive treatment primarily aims at controlling blood pressure (BP), but is also shown to control urine albumin excretion. This renoprotective role of antihypertensive medications consists of halting or reverting albuminuria progression. PATIENTS AND METHODS: A national Kingdom of Saudi Arabia (KSA), multicenter, observational, longitudinal study (RATIONAL), evaluated the correlation between BP control and microalbuminuria evolution over 1 year. Adult hypertensive patients with kidney damage were enrolled, after giving written consent. RESULTS: Of 409 patients, 60% had uncontrolled BP at baseline, down to 34% at 12 months. Over 80% of patients were on mono or double antihypertensive therapy, and angiotensin-receptor blockers (ARB) topped the list of medication classes. Albumin-creatinine ratio (ACR) significantly decreased throughout the study, indicating that BP control is paramount to prevent target organ damage. BP change most strongly correlated with ACR change upon triple therapy (ARB + calcium channel blocker + ß-blocker). Importantly, 25% (at 6 months) and 38% (at 12 months) of patients reverted back to normoalbuminuria, mostly upon renin-angiotensin system blockers. Around 80% of study patients had also diabetes, a common condition in KSA, which significantly hindered achievement of normoalbuminuria at 12 months. CONCLUSION: A modest but solid correlation between BP control and ACR reduction was identified. Results underline proper BP management in KSA and success of antihypertensive treatment in reverting microalbuminuria or delaying its progress. The study duration might be insufficient to reflect conclusively the beneficial effect of longer-term BP control on microalbuminuria evolution.

Effect of plasma progesterone on oocyte recovery, oocyte quality, and early in-vitro developmental competence of embryos in Bos indicus dairy cows.

The objective of present study was to determine the effect of plasma progesterone (P4) on oocyte recovery, oocyte quality, and early in-vitro developmental competence of embryos in Bos indicus dairy cows. The ovaries were collected in an abattoir. These ovaries (n = 750) were divided into two groups: 1) estrous CYCLIC (n = 318), and 2) estrous ACYCLIC (n = 432). Mean serum concentrations of P4 in a subset of (n = 85; 4.21 ± 0.4 ng/ml compared with 0.5 ± 0.2 ng/ml; P < 0.05) were greater in estrous CYCLIC as compared to ACYCLIC cows, respectively. The mean number of oocytes recovered per ovary (6.5 ± 0.5 compared with 4.0 ± 0.2; P < 0.05) was greater for estrous CYCLIC than ACYCLIC cows, respectively. The oocytes with grade I_+_II quality (55.3% compared with 47.6%; P < 0.05) were greater, whereas, there was lesser percentage with grade III_+_IV quality (44.5% compared with 52.4%; P < 0.05) from estrous CYCLIC as compared with ACYCLIC cows, respectively. Cleavage rate (70.9% compared with 52.8%; P < 0.05) was greater for embryos derived from estrous CYCLIC than ACYCLIC cows, respectively. Similarly, the embryo developmental rates to the 8- (38.5% compared with 20.8%; P < 0.05) and 16- (20.0% compared with 10.9%; P < 0.05) cell stage were greater for embryos derived from estrous CYCLIC as compared to ACYCLIC cows, respectively. In conclusion, the presence of greater plasma P4 has a beneficial effect on oocyte recovery, oocyte quality, and early IVEP outcomes in Bos indicus dairy cows.

Effect of oestrus synchronization methods on oestrus behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes.

The objective of this study was to determine the effect of oestrous synchronization methods on oestrous behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes. In Experiment 1, oestrous behaviour and timing of ovulation were determined from (n=34) oestruses. The mean (+/- S.E.M.) time of ovulation after the onset of standing oestrus was greater (P<0.05) in PGF(2alpha)-induced luteolysis (30.6+/-1.5h) compared to Ovsynch buffaloes (15.0+/-0.8h). In Experiment 2, pregnancy rates were compared between two methods of synchronization (detected oestrus and Ovsynch protocol) during the breeding and low breeding seasons. Pregnancy rates of buffaloes bred at detected oestrus (62.5%) or by the Ovsynch protocol (36.3%) during the breeding season did not differ significantly (P>0.05) from those which were inseminated during the low breeding season (55.5%) and (30.4%), respectively. This study demonstrates clearly that (1) timing of ovulation in Nili-Ravi buffalo is about 30h after the onset of standing oestrus and (2) buffaloes can be successfully synchronized with optimum fertility using either PGF(2alpha) alone (detected oestrus) or using (Ovsynch protocol) during low breeding season, to calve during the period when milk availability is short.