2. MATERIALS AND METHODS2.1. Animals and ManagementA total of sixty normal calving pluriparous buffaloes with live body weight ranged between 402 and 612 kg (507.14 ± 18.90 kg; mean ± SEM) and body condition score (BCS) of 2.9 ± 0.15 were included in this study. The animals belonged to buffalo farm in El-Badary city, Assiut province, Egypt (latitude: 8.2 meters, 25.4 centimetres, 58N; longitude 10.1 meters, 25.4 centimetres, 58E; altitude 37 m), kept in free-stall barns and fed ad libitum according to NRC [10]. The total mixed ration (TMR) included mainly alfalfa, corn silage, beet pulp, cotton seed, soyabean, corn and barley. All animals were apparently clinically healthy and free from external and internal parasites. The cows were machine-milked twice daily at 5.00 am and at 6 pm. The mean 305-day milk yield of the cows in the preceding lactation was 2592.80 kg the animals were lived under the same management and prevalent environmental conditions between October 2009 and February 2010.
2.2. Experimental DesignAll buffaloes were examined till the 75th day postpartum (p.p.) starting from 14th day p.p. on a weekly basis. During the examination, cows were examined by vaginoscope and transrectal ultrasonography (US) after normal parturition.
2.3. Body Condition Scoring and Animal WeightIn all animals Body Condition Score (BCS) was conducted at each examination based on a scale from 1 (emaciated) to 5 (very fat) [11]. The animals were weighted each time and recorded till further analysis.
2.4. Gynaecological and Transrectal Ultrasonographic ExaminationsTransrectal palpation was carried out to assess the position and degree of uterine involution. An attempt to monitor uterine involution was done by measuring the weekly variation of the transverse diameter of the uterine horns in ultrasonographic image. Since it was difficult to achieve consistent measurements, uterine involution was considered complete when the uterus was reduced in size, easily retractable, had normal tonicity and was completely located in the pelvic cavity. Reproductive tract measurements included uterine horn size at the base of the horn, ovarian status. The uterus was considered involuted when the difference between pregnant and non-pregnant horns became <10 mm, while non-involuted uterus when the difference between both horns was ≥10 mm [12]. The cervical diameter (CD), previous gravid uterine horn (PGUH) and non-gravid uterine horn (NPGUH) diameters were determined transrectally. The PGUH was differentiated from NPGUH where it was longer and of greater diameter than the NPGUH at the first examination (14th day) [12,13]. Ultrasonographic evaluation was performed using a 6/8 MHz rectal linear array transducer connected to B-mode ultrasound scanner (100 LC, Pie Medical, Masstricht, The Netherlands). Data obtained by ultrasonography included: diameter of the uterine horns (mm), number and size of follicles (≥5 mm), presence of luteal tissue (yes/no), and presence of fluid in the uterine horns (yes/no). The ultrasonographic equipment was supplied with image freeze and electronic calliper functions for taking measurements. Follicles were defined as nonechogenic rounded structures with a clear demarcation between the follicular wall and antrum. A corpus luteum (CL) was defined as a grainy echogenic structure that had a well-defined border with the less echogenic ovarian stroma, and in some corpora lutea, there was a nonechodense lacuna [14]. The maximum diameter of each structure was measured using the electronic calliper. The number of small follicle (≥5 mm diameter), dominant follicle (DF, ³8 mm diameter) and CL in both ovaries (ipsilateral and contralateral) to the previous gravid uterine horn was recorded.
2.5. Blood Sampling, Biochemical and Steroids AssaysBlood samples were collected from the jugular vein into heparinized vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ) at 14 - 28, 29 - 36, 37 - 44, 45 - 60 and 61 - 75 days postpartum for determination of plasma concentrations of Progesterone (P4), Estrogens (E2), triglyceride, cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDLC), very low density lipoprotein cholesterol (VLDL-C) and glucose. Progesterone was analyzed to assure the presence of luteal tissue which indicated resumption of reproductive cyclicity. Plasma estrogen (E2) and progesterone (P4) concentrations were determined using ELISA kits (Bio Check, Foster City, CA 94404, USA) using the micro-well method. The kit had a sensitivity of 10 pg/ml with the interand intra-run precision coefficient of variations of 6.4% and 4.1%, respectively for estrogen, while for progesterone, the sensitivity of 0.0625 ng/ml with the interand intra-run precision coefficient of variations of 4.5% and 2.6%. Glucose (Glu, gm/l), total cholesterol (C, mg/l) triglyceride (TG, mg/l) and highdensity lipoprotein cholesterol (HDL-C) were determined colorometrically using commercial test kits supplied and by Digital Ultraviolet Spectrophotometer (Digital Ultraviolet Spectrophotometer, CE 292, series 2, Cecil instruments, Cambridge England, Series No. 52.232). Lowdensity lipoprotein cholesterol (LDL-C) was calculated according to Friedewald et al. [15].
2.6. Animal Breeding and Pregnancy DiagnosisAnimals were bred after a voluntary waiting period of 70 - 85 days pp. They were bred after spontaneous normal estrus by natural mating using a good fertile buffalo bull. Pregnancy was diagnosed using transrectal ultrasonographic examination at 60 and 90 days post-service. Pregnancy status at 90 days was used for the analysis. Based on 90 days pregnancy diagnosis within 250 days pp, overall conception rate (OACR), interval from calving to first service (DFS), number of service per conception, and days open (DO) were calculated, and the animals retrospectively were allocated into pregnant group (PREG, animals that became pregnant at 90 days post estrus) and non-pregnant group (NPREG, animals that failed to become pregnant within 250 days pp).
2.7. Statistical AnalysisThe principal points of the study were predomination ovarian structures, uterine involution and blood metabolites as energy indicators in all animals starting from 14th to 75th day p.p. and the interaction of these factors on the reproductive performance. The general linear models ANOVA for repeated measures [16] was used for determining the main effects of each group and days, and their interaction. If a significant effect of time or a significant interaction was determined, a Fisher’s least significant differences (LSD) test was used as a post hoc analysis to locate mean differences among groups within days and among days within groups. Using Chi-square, outcomes measured to assess reproductive performance as overall conception rate (%) and Interval from calving to first estrus were compared. The diameter of previous gravid uterine horn (PGUH) and the uterine horn contralateral to it (NPGUH) and cervical diameters as well as number of small and dominant follicles and the proportion of buffaloes with follicles ³ 8 mm were compared in the two groups (pregnant and non-pregnant) using t-test. Level of significance was set at P < 0.05.
3. RESULTS3.1. Conception RateIn the present work, over all conception rate (OACR) was 34 animals of 60 (56.66%), while 26 animals (43.33%) were diagnosed as non pregnant.
3.2. Animal Weight and BCSBody weight and BCS presented in Table 1. There were significant differences between pregnant and non-pregnant groups in overall means of BCS and body weights during p.p. (P < 0.01). The differences in BCS and body weight at the periods of examinations between the two groups were not significant up to the 24th day p.p., thereafter there were significant differences up to 75th day p.p. (Figure 1).
3.3. Uterine Condition, Ovarian Findings and First p.p. Reproductive PerformanceThe data of uterine and ovarian findings were showed in Tables 1-3. There was a significant difference between PGUH and NPGUH diameter in pregnant, non-pregnant groups at 28th day postpartum. There were no significant
Table 1. The overall mean of post partum weight, BCS and uterine condition in buffaloes.
Table 2. The number of follicles ³ 5 mm diameter in the ovary ipsilateral or contralateral to the PGUH at 2 time periods post-partum.
Table 3. The percent of animals possessing follicles ³ 8 mm diameter (DF) and CL in the ovary ipsilateral or contralateral to the PGUH between 14 - 28 days postpartum.
Means with different superscript in the same row significant differ (a:b, c:d, P < 0.01).
differences in cervical diameter and the number of animals showing intrauterine fluid up to Day 28 p.p. between the two groups. The Interval from calving to first estrus in the pregnant group was shorter than that in non-pregnant group (Table 1).
Follicles (≥5 mm diameter) were observed in both ovaries ipsilateral and contralateral to the PGUH. There was no significant difference in number of those follicles between pregnant and non-pregnant animals. The number of the same follicles in the ovary ipsilateral to the PGUH was fewer (P < 0.01) than that in the ovary contralateral to the PGUH between 14 - 28 days p.p. (Table 2).
The proportion of buffaloes with follicles ³8 mm diameter in ipsilateral and contralateral ovary to the PGUH in pregnant animals was higher (P < 0.01) than that in the non-pregnant buffaloes. The number of buffaloes having CL in ipsilateral ovary to the PGUH in non-pregnant group was higher (P < 0.01) than that in the pregnant buffaloes. The number of corpora lutea in contralateral ovary to the PGUH in pregnant animals was higher (P < 0.01) than that in non-pregnant buffaloes (Table 3). The Interval from calving to first service in the pregnant buffaloes which had DF in the ovary ipsilateral to the PGUH (n = 16) was shorter (P < 0.01) than that having no dominant follicles (n = 18). The number of service per conception and days open in the pregnant buffaloes without DF in the ovary ipsilateral to the PGUH were higher and longer (P < 0.01) than in buffaloes group possessing DF (Table 4).
3.4. Plasma P4 and Estrogens ConcentrationsThe overall mean of P4 and E2 concentration during p.p. in pregnant and non-pregnant buffaloes showed in Table 5. There were significant differences between the two groups in overall mean of P4 and E2 concentrations. Significant differences in the P4 concentration were recorded between groups at 14 - 45 days p.p. Estrogen concentration showed significant differences between pregnant and non-pregnant at 29 - 45 days p.p. After 45th day, the differences in both P4 and E2 in the two groups were not clear (Figure 2).
3.5. Blood Metabolic and Biochemical ParametersOverall mean of Plasma Glucose, cholesterol, Triglyceride, lipoprotein cholesterol and steroid hormone in pregnant and non-pregnant buffaloes during the experiment was presented in Table 5. The concentrations of glucose and triglyceride were higher (P = 0.057) in pregnant than non-pregnant group. LDL-C and VLDL-C concentrations were higher (P < 0.01) in non-pregnant group than pregnant buffaloes. There were no significant differences between pregnant and non-pregnant buffaloes in overall mean concentrations of cholesterol and HDL-C.
The glucose level was higher (P < 0.05) in pregnant
Table 4. The postpartum reproductive performance in pregnant buffaloes with or without dominant follicles in the ovary ipsilateral to the PGUH between 14 - 28 days.
*DF/PGUH, dominant follicle in the ovary ipsilateral to previous gravid uterine horn.
buffaloes than non-pregnant buffaloes between 14th and 44th days p.p., then there was no significant difference (Figure 3). In pregnant group, the level of triglyceride was higher (P < 0.001) than non-pregnant one between 14th and 36th day p.p. and the highest concentration was recorded between 45th and 60th day (Figure 3).
The highest level of cholesterol was recorded in pregnant group between 14th and 45th day p.p. and the difference between pregnant and non-pregnant buffaloes was not significant. There were no significant differences in HDL-C concentrations (Figure 4).
The LDL-C level was higher (P < 0.001) in pregnant buffaloes than non-pregnant group between 29th and 75th days p.p. In non-pregnant group, the level of VLDL-C was higher (P < 0.01) between 14th and 44h day p.p., while was higher (P < 0.01) in pregnant group between 45th and 60th day p.p. (Figure 5).
4. DISCUSSIONThe objective of the current work was to uterine involution or resumption of estrous activity is likely to prolong the calving interval and reduce the lifetime reproductive and productive efficiency [1]. There were significant differences between pregnant and non-pregnant group in overall means of BCS and body weights during the experiment period p.p. The reported findings here are consistent with other studies, in cattle [17-19]. These results indicated that non-pregnant group suffered from NEB more than the pregnant one therefore, the loss in body weight and marked decrease in BCS between the third and tenth week p.p. associated with decrease in the
Table 5. Overall mean of biochemical parameters in pregnant and no pregnant buffaloes (Mean ± SEM).
conception rate. Earlier reports coincide with ours that body weight (BW) variation during postpartum period has an important role on reproductive performance; a more pronounced loss of BW was observed in the cows that did not resume ovarian activity compared to the cows with ovarian activity resumption within seven weeks postpartum, but the statistical analysis did not evidence differences [18,19]. It should be considered that in this trial, the most pronounced mean body weight loss (–6.46%) was less than values reported by Heinonen et al. [20] who observed lower reproductive performance in cows that lost more than 10% of BW postcalving compared with cows that lost less than 10%.
No significant differences were detected in the whole period of observation in the amount of milk production. These results are consistent with other studies that found no relationship between milk production and reproduction [21,22]. However, most of the recent studies have found a negative relationship between milk production and several fertility traits [23-27]. On the contrary, Buckley et al. [18], observed a positive association between milk yield variables and reproductive efficiency.
In the current work, the number of follicles ≥ 5 mm diameter in the ovary ipsilateral to the PGUH was fewer than that in the ovary contralateral to the PGUH between 14 - 28 days p.p. in both pregnant and non-pregnant buffaloes. In dairy cattle similar results was recorded during first 4 weeks p.p. [28,29]. Follicular activity started 6 days earlier in the ovaries contralateral to the gravid horn (21 days) than in the ipsilateral (27 days) ovaries [30] and was higher in the same ovaries during the first 35 postpartum days [31]. The explanation of this observation may be due to influence of the follicle on the uterine endometrium and/or myometrium. One hypothesis is that estradiol synthesized by a follicle ³ 10 mm diameter has a beneficial local effect on the uterine function [28]. Plasma estradiol concentrations are greater within the uteroovarian vein draining the ovary containing the ovulatory follicle [32]. Similarly, the correlation between the uterine involution and estradiol level was observed in this work and further study should be done for better documentation this point. The findings in the present study showed that the presence of dominant follicles ³ 10 mm diameter in the ovary ipsilateral to the PGUH between 14th and 28th Day postpartum is associated with shorter calving to conception interval and fewer number of services per conception. Our results agree with that recorded in cattle [28,29,33]. There was no significant difference in cervical diameter between the two groups at 28th day p.p. The cervical involution completed earlier than uterine involution in both groups and the differences were not significant, similar results were recorded, where the cervix was completed by 24 - 39 days postpartum [34-37].
It was reported that the cows which had an initial ovulation before, rather than after 41 days postpartum had shorter calving to conception intervals [38]. Similarly, Darwash et al. [39] reported a significant reduction in calving to conception interval accompanied with shorter intervals to the first post-partum increase in milk progesterone concentration, while Smith and Wallace [40] recorded the reverse. The current study showed that the proportion of buffaloes with ovarian activity as defined by ultrasonography for presence of CL and/or follicles ³ 8 mm diameter, had a shorter calving to conception interval and became pregnant. It was observed also that during the first 6 weeks p.p., the level of both E2 and P4 was higher in pregnant buffaloes than that in non-pregnant group. The difference may be due to the number of animals having DF on both ipsilateral and contralateral ovary and CL on contralateral ovary to the PGUH. The number of CL on ipsilateral ovary to the PGUH was greater in non-pregnant than in pregnant group, this may be due to CL of previous pregnancy retained longer in non-pregnant group. Similar results were reported in buffaloes [30] and in dairy cows [29].
Many factors affecting the initiation of follicular activity during p.p. period in buffaloes were studied; suckling [41,42], level of milk production [31] and prepartum nutrition [42]. It was recorded that suckling; milk production and prepartum nutrition did not influence the interval to initiation of follicular activity [31,41,43]. However, the relationship between milk production, nutrition and reproductive performance couldn’t record clearly because there were no differences in the milk quantity and nutrition between the tested groups.
Earlier studies reported variable length of days open as 40 to 228 days. In non-suckled buffaloes the interval from calving to conception was reported as 126 days in Murrah [44], 168 days in Nili-Ravi [45] and 104 days in Surti breed [46]. These greatly varying intervals reported by different workers could be due to breed, nutritional status, management, soil type, weather condition and even postpartum breeding policies adopted in different areas of the country. In present work, the Interval from calving to first service in the pregnant group was shorter than that in non-pregnant group, due to earlier ovarian resumption in the pregnant than in non-pregnant group. The pregnant cows with high genetic merits showed higher days open and services per conception, even though with an earlier recovery of cyclicity. Furthermore, non pregnant with high genetic merits group, with similar energy deficiency, showed more subclinical health problems [47]. Our results were in agreement with that reported in surti buffaloes where the occurrence of first postpartum oestrus, service period and calving interval were significantly shorter/lesser in fertile group of buffaloes than the infertile one [48].
The levels of glucose fluctuated significantly during different weeks postpartum and the difference between pregnant and non-pregnant buffaloes was significant. It could be due to a high degree of negative energy balance that may affect the subsequent fertility. Similar results were reported in cows [47] but there were no differences between fertile and infertile buffaloes [48].
It is well established that LDL-cholesterol (LDL-C) is one of the most important parameters for estimating hypercholesterolaemia. Although bovine plasma apoB-100 is useful for diagnosis of fatty liver and related diseases in dairy cows [49-52], the clinical significance of bovine LDL-C is unknown. A decrease of apoB-100 also occurs in cows during the early lactation stage [53,54], which is probably caused by hepatic TG accumulation. In this study, significant depletion of LDL-C was observed in cows at parturition and which not became pregnant. It is well established that the most important role of LDL is delivery of cholesterol to peripheral tissues via LDL receptors. Rudling and Peterson [55] clearly demonstrated that the tissue concentrations of LDLs receptors are highest in corpus luteum or adrenal cortex, and that plasma LDLs are closely related to tissue LDL receptors. On the other hand, it was reported that there was no difference in the ability of LDL-C and HDL-C to stimulate progesterone production by cultured bovine corpus luteum cells [56]. The clinical importance of bovine serum LDL-C for steroidogenesis remains unclear. However, explanation and validation require further targeted cohort investigation to find reasons for later resumption of ovarian activity as well as effect of biochemical on reproductive performance in buffaloes.