Endometritis impairs luteal development, function, and nitric oxide and ascorbic acid concentrations in buffalo (Bubalus bubalis)

A vast majority of the world buffalo resource is concentrated in tropical and subtropical countries. Apart from heat stress and poor nutritional availability, endometritis is one of the most commonly encountered reproductive problems limiting fertility and consequently productive potential of the species. As demonstrated recently, endometritis impairs growth and follicular fluid composition of the largest follicle in buffalo. In the present study, the effect of endometritis on luteal development, function, nitric oxide (NO), and ascorbic acid was investigated. Reproductive tracts were collected from 90 cyclic buffaloes at an abattoir and grouped into endometritic (n?=?36) or non-endometritic (n?=?54) buffaloes based on physical examination of uterine mucus, white side test, and uterine cytology. Samples with pus-containing mucus, positive reaction on white side test, and/or >5 % neutrophils were considered to be positive for endometritis. Corpora lutea were enucleated, weighed, classified into stages I to IV, and assayed for progesterone (P₄), NO, and ascorbic acid concentrations. Endometritic buffaloes had lesser (P?<?0.0001) luteal weight and P₄, NO, and ascorbic acid concentrations than non-endometritic buffaloes. The findings indicated that endometritis impairs corpus luteum development and function in buffalo. Reduced luteal NO and ascorbic acid concentrations during endometritis are novel findings.     

Relationship between endometritis and oxidative stress in the follicular fluid and luteal function in the buffalo

In this study, alteration in the follicular fluid composition and luteal function was investigated in the buffalo with endometritis. Genitalia were classified into cytological and purulent endometritis on the basis of polymorphonuclear cell cut off while non‐endometritis served as control (n = 10/group). In the follicular phase, the number of surface follicles was counted, diameter of the largest follicle was measured and the follicular fluid was assayed for total protein, cholesterol, malondialdehyde (MDA), total antioxidant capacity (TAC), oestradiol (E₂) and progesterone (P₄). The P₄ content of corpus luteum during mid‐luteal phase was estimated by radioimmunoassay. Ovaries from the follicular phase of oestrous cycle showed no significant difference in the total number of surface follicles, size of the largest follicle and volume of follicular fluid in the buffaloes with and without endometritis (p > .05). However, the antral fluid of the largest follicle from the genitalia of buffalo with cytological and purulent endometritis showed a significant decrease in the concentration of total protein, cholesterol, TAC and E₂ and a significant increase in the concentration of MDA and P₄ (p < .05). The results indicated that there is an association between endometritis and decreased ovarian function.     

Biochemical analysis of uterine fluid for identification of indicators for subclinical endometritis in the water buffalo (Bubalus bubalis)

Alterations in biochemical constituents of uterine fluid have been suggested for diagnosis of subclinical uterine infection in the bovine. This study was undertaken to investigate whether uterine fluid biomolecules could act as tool for diagnosis of subclinical endometritis in the buffalo. Uterine fluid samples from normal (n = 22) and subclinical endometritis (n = 18; diagnosed based on uterine cytology)‐affected buffaloes were subjected to biochemical analysis. Among the different biochemical constituents estimated, urea, urea N, cholesterol, total bilirubin and alkaline phosphatase (ALP) concentrations were significantly (p < 0.05) higher in uterine fluid obtained from subclinical endometritis‐affected buffaloes. The extent of difference between normal and subclinical endometritis‐affected buffaloes was highest in ALP (69%) followed by cholesterol (55%), bilirubin (48%), urea (30%) and urea N (30%) concentrations. Receiver operating characteristic (ROC) analysis indicated that the likelihood ratio (LR) was 3.63 for urea, indicating that buffaloes having less than the threshold concentration (47.5 mg/dl) of urea in their uterine fluid were at 3.6 times more risk to be affected with SE. The LRs for urea N, cholesterol, ALP and bilirubin were 2.33, 2.54, 2.12 and 1.65, respectively. It was concluded that ALP, urea, urea N and cholesterol concentrations in uterine fluid may serve as an aid for diagnosing subclinical endometritis in the buffalo.     

A Study of Endometritis Causing Repeat Breeding of Cycling Iraqi Buffalo Cows

The objectives of this study were to determine the non‐specific aerobic and anaerobic bacterial causes of endometritis causing repeat breeding of cycling Iraqi buffalo cows at Nineveh province, validate diagnostic criteria for endometritis and to evaluate the treatment efficiency of using systemic or intra‐uterine infusion of antibiotics for the treatment of endometritis. Data were collected from 60 buffalo cows with history of repeat breeding in different herds. All buffaloes were subjected to detailed clinical examination including external inspection, vaginoscopy and transrectal palpation of the cervix, uterus and ovaries. Swabs for bacteriology and biopsies for histopathology were collected from the uterine lumen from each cow. Character, odour and estimation of polymorphonuclear cells (PMN) of the vaginal mucus were scored. Blood samples were collected from cows for creatine kinase (CK) and aspartate aminotransferase (AST) measurement. Treatment conducted using oxytetracycline with tylosin in local intrauterine infusion or systemically with hormonal treatment. The most pre‐disposing factor for uterine infection was retained placenta (13.3%). The most prevalent bacteria in uterine lumen were E. coli (23%), Archanobacterium pyogenes (13%) and Staphylococcus aureus (10%) were mostly isolated from buffaloes with repeat breeding. Vaginal mucus character score was associated with the bacterial growth density score. The difference in PMN was highly significant (p < 0.01) in animals with repeat breeding than control groups. In addition, PMNs was significantly (p < 0.01) correlated r = 0.894 with the character of vaginal discharge. High level of PMNs observed in buffaloes infected with A. pyogenes. Buffalo cows with endometritis had higher CK (321.47 ± 39.06 vs 162.01 ± 16.41 U/l) and AST (133.93 ± 12.43 vs 97.01 ± 6.86 U/l) activities (p < 0.05) than control‐heifers, but no significant difference was observed between buffalo cows with endometritis in CK (321.47 ± 39.06 vs 208.33 ± 5.84) and AST (133.93 ± 12.43 vs 156.17 ± 9.65) activities than control‐pluriparious. It could be concluded that A. pyogenes was the only non‐specific uterine pathogen directly associated with severe endometrial lesions. Vaginoscopy examination combined with palpation of uterus increase the accuracy of diagnosing endometritis and cytogenic examination of uterine discharge is more reliable method of establishing the presence or absence of uterine inflammation in buffalo cows. Animals with repeat breeding (endometritis) showed clinical cure and improved pregnancy in all treatment groups with no significant difference. The use of oestradiol in repeat breeder cases has no effect in improving neither clinical cure rate nor pregnancy rate.     

Ovarian Follicular Dynamics in Buffalo Cows (Bubalus bubalis)

Follicular growth in Egyptian buffalo cows was monitored using genital tracts from 200 buffalo cows collected immediately after slaughter. According to the morphological appearance of the corpus luteum (CL), the corresponding oestrous cycle was divided into four stages: A (days 1–4), B (days 5–10), C (days 11–17) and D (days 18–21). Within these stages the follicular population on the ovaries was evaluated and the dominant follicle (DF) determined in all recovered ovaries. The functional status of the DF and the largest sub‐dominant follicles was examined by histological examination in 31 cases, and Radio Immunoassay (RIA) analyses for estradiol‐17β (E₂) and progesterone (P₄) was performed in the follicular fluid in 23 of the DF. The results showed that DFs changed their endocrine character within the stages of the oestrous cycle. The DFs between days 5 and 10 were functionally active (E₂‐dominant; non‐atretic) in most of the cases. Between days 11 and day 17 half of the DFs became functionally inactive (P₄‐dominant; atretic). At days 18–21 all of the DF became functionally active and non‐atretic. In the specimens that carried two large follicles one of them was regularly atretic and P₄‐dominant whereas the other was non‐atretic and E₂‐dominant. Between days 18 and 21 all ovaries examined showed at least one large follicle. These findings suggest that in most of the cases follicular dynamics occurs in two wave‐like patterns in the Egyptian buffalo cows.     

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.     

Follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in water buffalo (Bubalus bubalis)

The objective of this study was to examine the follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in buffaloes. Ovaries were collected from 56 acyclic and 95 cyclic buffaloes at slaughter, surface follicle number was counted and follicles were classified into small (5.0–6.9 mm), medium (7.0–9.9 mm), and large (≥10.0 mm) size categories based on their diameter. Follicular fluid was aspirated and assayed for nitric oxide, ascorbic acid, estradiol, and progesterone. Acyclic buffaloes had a higher (P < 0.05) number of medium-sized follicles and a lower (P < 0.001) number of large follicles than the cyclic ones. In acyclic animals, the number of large follicles was lower (P < 0.01) than in medium size category which in turn was lower (P < 0.001) than the number of small follicles. In contrast, the number of medium and large follicles was not different (P > 0.05) in the cyclic control. However, the number of small-sized follicles was higher (P < 0.001) compared to the other two categories. The incidence of large-sized follicles was lower (P < 0.05) in acyclic buffalo population compared to the cyclic control. Evaluation of estrogenic status demonstrated that all the follicles of acyclic buffaloes are estrogen-inactive (E ₂/P ₄ ratio < 1). Small- and medium-sized follicles of acyclic buffaloes had higher concentrations of nitric oxide (P < 0.05 and P < 0.001, respectively) and lower concentrations of ascorbic acid (P < 0.05 and P < 0.01, respectively) than the corresponding size estrogen-active follicles of their cyclic counterparts. In conclusion, this study indicates that follicular development continues during acyclicity in buffaloes. Although follicles in some acyclic buffaloes attain a size corresponding to morphological dominance, they are unable to achieve functional dominance, perhaps due to an altered balance of intrafollicular nitric oxide and ascorbic acid and, as a result, these follicles instead of progressing to ovulation undergo atresia.     

Follicular dynamics and colour Doppler vascularity evaluations of follicles and corpus luteum in relation to plasma progesterone during the oestrous cycle of Surti buffaloes

With an objective to evaluate the follicular dynamics and vascularity changes in follicles and corpus luteum, the ovaries of cyclic Surti buffaloes (n = 9) were examined daily sequentially by transrectal B‐mode and colour flow mode (CFM) ultrasonography starting from the day of oestrus till the onset of next oestrus. Higher proportion of buffaloes evidenced one‐wave cycle (66.66%) compared to two‐wave cycle (33.34%) with none showing a three‐wave cycle. The dominant follicle of the first follicular wave was the ovulatory follicle and persisted for 19.70 ± 0.50 days compared to its persistence for 16.5 ± 1.45 days in a two‐wave cycle. The maximum diameter of the ovulatory follicle in a one‐wave and two‐wave cycle did not differ yet their linear growth rates were significantly lower (p < 0.01) in a one‐wave cycle. Colour flow mode examination of follicles revealed that the percentage of follicles with detectable blood flow in the subsequently determined largest follicle (dominant follicle) was not different from that in the second largest follicle before follicle deviation. The blood flow in the dominant follicle increased significantly on the day of oestrus. The mean diameter and blood flow to the corpus luteum (CL) increased linearly and significantly from Day 5 of oestrus till Day 13 after which both parameters started declining. At or around Day 16, there was precipitous fall in the blood supply to the CL and CL diameter that continued declining thereafter to reach the lowest around Day 20 of the oestrous cycle. Rise in plasma progesterone concentrations was synchronous to CL diameter and vascularity and showed significant and positive correlations. It was concluded that Surti buffaloes evidence a preponderance of one‐wave follicular growth pattern with a significant increase in the vascularity of ovulatory follicle on the day of oestrus and corpus luteum on Day 13 of the oestrous cycle.     

Dominant follicles development and estradiol‐17β concentrations in non‐ovulating and ovulating post‐partum Bulgarian Murrah buffaloes

This study was designed to evaluate the dominant follicles development and the estradiol‐17β concentrations in non‐ovulating and ovulating post‐partum buffaloes. Sixteen Bulgarian Murrah buffaloes were submitted to transrectal ultrasonographic examination from the 1st post‐partum day until day 50, 3 days apart. The follicular diameter of the different categories of follicles and the ovulations was recorded. The animals were allocated into two groups: I (n = 6) non‐ovulating and II (n = 10) ovulating buffaloes. Serum estradiol‐17β concentrations on the days for dominant follicle registration were measured by enzyme‐linked immunosorbent assay. The results were statistically processed by analysis of variance, non‐parametric and correlation analysis. The mean intervals between calving and first dominant follicle detection differed significantly (p < .05) among the groups (19.5 ± 6.2 vs. 13.8 ± 5.1 days), while the mean intervals between registered dominant follicles from two successive waves were comparable. The mean follicular diameters for the same category follicles in both groups were similar. Different estradiol‐17β concentrations (p < .05) for the first dominant follicle between non‐ovulating (23.5 ± 7.0 pg/ml) and ovulating (33.3 ± 8.4 pg/ml) buffaloes were determined. The cumulative percentages of buffaloes with firstly detected dominant follicle and ovulating animals correlated positively (r ≥ .84; p < .05) to post‐partum days. In conclusion, non‐ovulating and ovulating post‐partum Bulgarian Murrah buffaloes showed differences in the development of the first dominant follicle and estradiol‐17β concentrations during the time of dominant follicles detection.     

Intrafollicular and systemic serotonin,oestradiol and progesterone concentrations in cycling mares

The hypothesis that a local serotonergic network might also exist in the follicle of mares remains poorly documented, with exception for humans and laboratory species. For this reason, the aim of the present study was to clarify this possibility, investigating intrafollicular serotonin concentrations of the cycling mare at ovulation time. Sixty ovaries collected from 30 clinically healthy mares of slaughterhouse meat production with clinically normal reproductive tracts after slaughtering were evaluated. Blood samples were taken prior to sacrifice. Follicles were classified in three categories in relation to size, as small (20–30 mm), medium (31–40 mm) and large (>41 mm), and the follicular fluid samples were extracted from each follicle. Intrafollicular and systemic serotonin (5‐HT), oestradiol‐17β (E₂) and progesterone (P₄) were determined by means of enzyme‐linked immunosorbent assay and RIA, respectively. Intrafollicular 5‐HT, E₂ and P₄ concentrations were higher than systemic ones (p < .05). 5‐HT concentrations increased in larger compared to medium follicles, without differences compared to small size follicles (p < .05). 5‐HT and E₂ (r = .79) and 5‐HT and P₄ (r = .79; p < .05) were positively correlated. 5‐HT and P₄ concentrations in follicular fluid increased progressively with the increase in follicular size (p < .05). Follicle diameter and E₂ (r = .85) and P₄ (r = .68) were correlated (p < .05). Since serotonin interacts with steroids, its role on steroidogenesis during growth of the dominant follicle may be suggested.     

水牛有腔卵泡颗粒细胞凋亡的生物形态学特征

为了解水牛有腔卵泡闭锁的起因和有腔卵泡中是否存在卵泡颗粒细胞凋亡，本研究选择青春期前水牛和成年（发情间期和发情期）水牛的卵巢，采用石蜡组织切片、HE染色技术进行光镜观察，并采用DNA原位末端标记（TUNEL）技术和透射电镜观察研究了卵泡颗粒细胞凋亡的特征。结果表明：青春期前水牛的健康有腔卵泡上无或仅有极个别凋亡的卵泡颗粒细胞（GCs），而闭锁有腔卵泡上存在有多量凋亡的GCs，凋亡的形式：GCs层内出现单个或多个凋亡细胞，卵泡腔内出现凋亡小体群。在成年水牛发情间期的闭锁大卵泡上，GCs层弯曲皱折，存在多量凋亡的GCs。发情期水牛成熟卵泡的GCs层也存在GCs凋亡，排卵前的GCs层细胞全部脱落到卵泡腔内。本研究首次在同一组织切片上先后分别应用HE染色和TUNEL检查细胞凋亡，且证实了水牛有腔卵泡GCs凋亡的存在。超微结构显示了GCs凋亡的核边集化、核浓缩及凋亡小体的形态。上述结果提示：在水牛闭锁有腔卵泡中存在着GCs凋亡，GCs凋亡是启动水牛有腔卵泡闭锁的潜在机理。     

Correlation of Blood Flow of the Preovulatory Follicle to its Diameter and Endocrine Profile in Dairy Buffalo

Blood flow of the preovulatory follicle (POF) wall can be used as a predictor of the quality of POF. Our aim was to determine the correlation of blood flow of POF with the POF diameter, and intra‐follicular and plasma concentrations of Insulin‐like Growth Factor‐I (IGF‐1) and oestradiol in dairy buffalo. Nine Murrah buffalo subjected to an ovulation synchronization protocol (Ovsynch) were assessed on day 10 of the protocol for diameter and blood flow of POF, followed by the aspiration of follicle fluid. Prior to follicular aspiration, blood samples were obtained from jugular vein for estimation of IGF‐1 and oestradiol. The vascularity of POF was determined (Range: 250–967 pixel²) along with intra‐follicular and plasma concentration of IGF‐1 (Range: 9.3–31.8 ng/ml and 14.7–29.7 ng/ml respectively) and oestradiol (Range: 124.2–447.9 ng/ml and 0.25–1.05 ng/ml respectively). Diameter of the POF was weakly correlated (r = 0.21, p < 0.01) with blood flow to it. As compared to POF diameter, the blood flow of POF had greater positive correlation with intra‐follicular and plasma concentrations of hormones (IGF‐1 and oestradiol). A strong positive correlation was recorded between intra‐follicular IGF‐1 and oestradiol. Also, plasma concentrations of oestradiol and progesterone were negatively correlated In brief, assessment of the blood flow of the POF is a non‐invasive and reliable indicator of its functional competence as compared to the POF diameter.     

Effect of pre‐partum supplementation of vitamin E to Murrah buffaloes on immune functions and viability of calves

This study was undertaken in buffalo neonates born to vitamin E (dl ‐ alpha ‐ tocopherol acetate) ‐ supplemented and non‐supplemented Murrah buffaloes. Calves from vitamin E‐supplemented buffaloes (n = 10; vitamin E ‐supplemented calves [VeC]) and non‐supplemented buffaloes (n = 10; control calves [CC]) constituted the treatment and control groups respectively. Two colostrum samples were taken at the first post‐partum milking and again after 12 h from dams for IgG estimation. Sampling of blood was performed on days 0 (before colostrum feeding), 1, 3, 7, 14, 21, 28, 42, 56, 70, 84, 98, 112 and 126 post‐birth and analysed for apparent efficiency of absorption (%) of IgG and various immune parameters. Colostral IgG level was significantly higher (p < 0.05) in vitamin E‐supplemented buffaloes. The calves in both groups were born hypogammaglobulinemic with IgG level <5 g/l. However, first colostrum feeding resulted in significantly elevated IgG levels (>10 g/l) in calves of both groups at 24 h, which remained high afterwards. Apparent efficiency of absorption (%) of IgG at 24 h was significantly higher (p < 0.05) in VeC than in CC. Plasma Nitric Oxide (NO) levels were significantly elevated in the calves of either group at birth, which declined significantly (p < 0.01) afterwards. Vitamin E feeding to dams had no added effect on NO levels in experimental calves. Total leucocyte counts did not differ significantly between the two groups. However, lymphocyte and neutrophil counts changed significantly between groups (p < 0.01) and days (p < 0.01), with lymphocytes increasing and neutrophils declining with age. This study revealed that the calves were immunologically immature at birth. Ante‐partum supplementation of vitamin E did not influence plasma NO or IgG but had a significant effect on colostral IgG (p < 0.05). It also improved the apparent efficiency of absorption (%) of IgG at 24 h in VeC as compared to CC.     

A Study on Postpartum Metritis in Iraqi Buffalo Cows: Bacterial Causes and Treatment

The objectives of the present study were to determine the relationship between bacteriological findings, clinical signs and histopathological changes in postpartum metritis. Evaluation of the treatment efficiency of using systemic or intra‐uterine infusion of antibiotics with some hormonal preparations for the treatment of postpartum metritis. Data were collected from 50 buffalo cows with history of calving of more than 1 month. All buffaloes were subjected to detailed clinical examination including external inspection, vaginoscopy and transrectal palpation of the cervix, uterus and ovaries. Swabs for bacteriology and biopsies for histopathology were collected from uterine lumen from each buffalo included in the present study. Bacteria identified using API systems following aerobic and anaerobic cultures. Vaginal mucus scored for character, odour and estimation of polymorphonuclear cells (PMNs). Treatment conducted using oxytetracycline in local intrauterine infusion or systemically with hormonal treatment including prostaglandinF2α (PGF2α) and oestradiol benzoate. Results revealed that the most predisposing factor for postpartum uterine infection was retained placenta and toxic puerperal metritis. The most prevalent bacteria in uterine lumen were Escherichia coli, Archanobacterium pyogenes, Bacteroides fragilis and Fusobacterium necrophorum the most prevalent bacteria in buffaloes with postpartum metritis. A. pyogenes and F. necrophorum were an important pathogens causing severe uterine inflammation as found in histopathological examinations. Buffaloes with postpartum metritis showed good clinical cure when oxytetracycline injected systemically with PGF2α. Intrauterine infusion of oxytetracycline had no advantage for the treatment of uterine infection in buffalo cows with postpartum metritis. PGF2α improved clinical cure of buffaloes with postpartum metritis.     

Ultrasonographic and Endocrine Characterization of Follicular Waves in Llamas with a Special Reference to the Overlapping Phenomenon During Successive Waves

Ovarian follicular growth in non‐mated llamas occurs in successive waves that generally superimpose their origin on the regression of the preceding wave (overlapping), originating prolonged sexual receptivity in the species. The aim of this study was to perform an ultrasonographic and endocrine characterization of individual and successive waves in non‐mated llamas with a special interest on the overlapping phenomenon. Twelve llamas were examined daily by transrectal ultrasonography for at least two consecutive waves. In six females, blood samples were collected daily at the end of each examination. The development of the largest follicle (F) showed a wavelike pattern with a mean duration of 25 days. All waves evaluated were partially overlapped on the preceding wave and emerged at a mean interval of 15.8 ± 0.5 days. This interwave interval determines a mean overlapping degree of 32% of the wave length. Similarly, mean plasma oestradiol‐17β (E₂) concentrations followed a wavelike pattern. However, E₂ concentrations started to decline before the structural regression of the F was observed. Mean basal E₂ concentrations remain higher than 10.9 ± 0.6 pmol/l. In conclusion, follicular activity in non‐mated llamas is characterized by continuous emergence of successive waves that always overlap the preceding wave with variable degrees. E₂ production during the follicular wave is shorter in duration than the morphological development of the F. Finally, the overlapping phenomenon maintains increased plasma E₂ concentrations persistently and this could explain the prolonged periods of sexual receptivity registered in llamas.     

A study on the ovarian follicular dynamic in Iraqi Northern Buffaloes

The aim of the present study was to test the hypothesis of wave pattern of follicular growth and to monitor the ovarian follicular dynamic in Iraqi buffalo cows. Reproductive tracts were collected at random intervals slaughtered at Mosul abattoir. According to morphological appearance of the corpus luteum, the estrous cycle was divided into four stages. The number of subordinate follicle (<5 mm in diameter) was higher during stage 1 (metestrous) and stage (proestrous and estrous) than during other stages of the estrous cycles, 13.5 ± 6.08 and 4 9.41 ± 3.94, respectively. There were fewer follicles (5–8 mm in diameter) during early diestrous and proestrous, 1.66 ± 1.42 and estrous, 0.69 ± 0.47 than during metestrous, 4.53 ± 3.23 and late diestrous, 3.66 ± 2.23. Follicles > 8–12 mm in diameter were more numerous during early diestrous, 1.62 ± 1.29 and late diestrous, 1.03 ± 0.72. A total 38 (64.6%; 82/127) animals examined showed follicles larger than 8 mm during early and late diestrous (stage 2 and stage 3). This indicated that these buffaloes developed two follicular waves in their cycle. Buffaloes did not show follicles larger than 8 mm during early and late diestrous were 45 animals (35.4%; 45/127), but all of these presented one large follicle during the following stage. These buffaloes develop only one follicular wave in their cycle. It could be concluded that, 64.6% of Iraqi buffalo cows develop two patterns of follicular waves, and 35.4% showed one wave of follicular dynamics.     

Follicle Turnover and Pregnancy Rates Following Oestrus Synchronization Protocols in Mediterranean Italian Buffaloes (Bubalus bubalis)

An ultrasound assessment of follicle turnover following two different protocols for synchronization of oestrus and ovulation, as well as an assessment of achieved synchronization between ovulation and AI and conception rates in nulliparous and pluriparous buffaloes were carried out during months of increasing day length. Nulliparous buffaloes (n = 30) were subjected only to Ovsynch protocol whereas pluriparous buffaloes (n = 31) were assigned to Ovsynch (n = 14) or to PRID-pregnant mare serum gonadotrophin (PMSG) (n = 17) protocol according to the presence of functional CL confirming cyclic and acyclic conditions. Ultrasound examination of ovarian follicular dynamics at critical days in the course of synchronization treatments was employed to monitor the fate of the largest available follicles at the beginning of treatments. Such available dominant follicle would persist throughout the protocol as ovulating follicle (no-follicle shift) or would regress giving way to a new follicle to become dominant and ovulate (follicle shift). Furthermore, ultrasound monitoring would determine the degree of synchronization of ovulation and final outcome represented by pregnancy rates. Pregnancy rate following Ovsynch protocol was 40% (12/30) and 42.8% (6/14) in nulliparous and pluriparous buffaloes respectively (p = 0.8575). Most ovulations were synchronized and recorded at AI and the following day in nulliparous (24/30; 80%) and pluriparous (12/14; 85.7%) buffaloes respectively (p = 1.000). A follicle shift was recorded in 14 of 30 (46.6%) and 11 of 14 (78.5%) in nulliparous and pluriparous buffaloes respectively (p = 0.0466). Among established pregnancies: eight derived from follicle shift (66.6%) and four from no-follicle shift (33.3%) in nulliparous buffaloes, p = 0.0729 whereas in pluriparous buffaloes five (83.3%) derived from follicle shift and one from no-follicle shift (16.6%), p = 0.6154. Collectively, from 18 pregnancies in nulliparous and pluriparous buffaloes following Ovsynch protocol, 13 derived from follicle shift (72.2%) and five from no-follicle shift (27.7%), p = 0.0860. Pregnancy rate in pluriparous buffaloes following PRID-PMSG protocol was 70.5% (12/17). The majority of ovulations were synchronized and recorded at first, second AI and following day (13/17; 76.4%). A follicle shift occurred in 15/17 buffaloes (88.2%) and among the 12 recorded pregnancies, 11 derived from follicle shift (91.6%), p = 0.5147. In conclusion, pregnancy rates following Ovsynch protocol were similar in nulliparous and pluriparous cyclic buffaloes. A progestagen treatment on acyclic buffaloes but still displaying some ovarian follicular dynamics, resulted in significantly higher pregnancy rate compared with Ovsynch (p = 0.0376). According to the time of scheduled AI, a high degree of synchronized ovulations were recorded following the implementation of both protocols.     

Effect of Oviductal Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah Buffaloes*

The present study was undertaken to elucidate the effect of non‐luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non‐luteal based on ovarian morphology. Non‐luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10 000 g at 4°C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at ?20°C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris–citrate–egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non‐luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5°C, filled in 0.5 ml French straws, exposed to LN₂ vapour, plunged into LN₂ and then stored at ?196°C. The equilibrated and frozen–thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo‐osmotic sperm swelling test (HOST). In frozen–thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non‐luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze‐thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen–thawed semen. It was inferred that incorporation of non‐luteal whole oviductal fluid proteins improved the sperm quality in frozen–thawed semen in Murrah buffaloes.     

Impact of norgestomet supplementation during early luteal phase on subsequent luteal profiles and conception rate in buffalo: a preliminary study

The current study was aimed to establish the impact of progesterone supplementation (norgestomet progestagen) between days 4 to 10 post-ovulation on subsequent luteal profile and conception rate in buffaloes. The 28 Murrah buffaloes of second to fourth parity, having normal reproductive organs, were estrus synchronized by double PGF_2α protocol at 11 days apart. The buffaloes were inseminated during mid- to late estrus and thereafter repeated at 24 h interval. The buffaloes were randomly assigned into two groups: (1) control (no treatment, n?=?14) and (2) treatment group (CRESTAR ear implant, n?=?14). The CRESTAR ear implant (3 mg, norgestomet progestagen) was inserted subcutaneous between days 4 to 10 post-ovulation. The ovaries were scanned at estrus and thereafter on days 4, 10, 16, 21, and 40 post-ovulation to examine the preovulatory follicle (POF) and corpus luteum (CL) diameter. Each ultasonography was followed by blood sample collection for analysis of plasma progesterone concentrations following ovulation. The conception rate was similar (p?>?0.05) between treated and control buffaloes. The pregnant buffalo of the control group had larger (p?<?0.05) POF diameter than nonpregnant counterparts. The CL diameter was similar (p?>?0.05) in both treated and untreated control as well as in their pregnant and nonpregnant buffaloes of the respective groups. The plasma progesterone concentrations were higher (p?<?0.05) in the treatment group on the day 10 post-ovulation as compared to the control buffaloes. It is concluded that norgestomet supplementation had no impact on conception rate and CL diameter but enhances the plasma progesterone concentrations following treatment in buffaloes.