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Anand Agricultural University, Anand
Anand Agricultural University (AAU) was established in 2004 at Anand with the support of the Government of Gujarat, Act No.(Guj 5 of 2004) dated April 29, 2004. Caved out of the erstwhile Gujarat Agricultural University (GAU), the dream institution of Sardar Vallabhbhai Patel and Dr. K. M. Munshi, the AAU was set up to provide support to the farming community in three facets namely education, research and extension activities in Agriculture, Horticulture Engineering, product Processing and Home Science. At present there seven Colleges, seventeen Research Centers and six Extension Education Institute working in nine districts of Gujarat namely Ahmedabad, Anand, Dahod, Kheda, Panchmahal, Vadodara, Mahisagar, Botad and Chhotaudepur
AAU's activities have expanded to span newer commodity sectors such as soil health card, bio-diesel, medicinal plants apart from the mandatory ones like rice, maize, tobacco, vegetable crops, fruit crops, forage crops, animal breeding, nutrition and dairy products etc. the core of AAU's operating philosophy however, continues to create the partnership between the rural people and committed academic as the basic for sustainable rural development. In pursuing its various programmes AAU's overall mission is to promote sustainable growth and economic independence in rural society. AAU aims to do this through education, research and extension education. Thus, AAU works towards the empowerment of the farmers.
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ThesisItem Open Access ULTRASONOGRAPHY IN FOLLICULAR DYNAMICS, FERTILITY MANAGEMENT AND EARLY PREGNANCY DIAGNOSIS IN CATTLE(AAU, Anand, 2014) HADIYA, KAMLESHBHAI KHODABHAI; Dhami, A. J.These investigations were undertaken on 60 Gir and HF x K Crossbred cattle of University farm in Anand from January to June 2014 with the objectives to study the follicular dynamics of spontaneous as well as hormonally (Mid-cycle PG, Ovsynch and CIDR) induced estrous cycles and even during early pregnancy in postpartum cows and postpubertal heifers using real time B-mode transrectal ultrasound scanning; to detect early pregnancy and embryonic mortality using clinical diagnosis and ultrasonography; to evaluate the efficacy of estrus synchronization protocols toward fertility improvement in subfertile cows; to monitor blood biochemical and plasma endocrine profile of spontaneous and induced cycles during treatment and thereafter at weekly interval until next estrus or 42 days of pregnancy; and to correlate plasma progesterone and estradiol profile with ovarian changes during estrous cycle and early pregnancy. The normal cyclic (24) and infertile (36) animals were thoroughly screened for their genital health through gynaeco-clinical examinations. They were subjected to transrectal ultrasonographic scanning to study the follicular dynamics during various reproductive phases and during estrus synchronization treatments. The animals found in spontaneous or induced estrus were inseminated with good quality frozen-thawed semen. Pregnancy was confirmed in non-return cases on day 23, 28, 35 and 42 using transrectal ultrasonography and per rectal palpation 60 days post-AI. Infertile animals (subestrus and repeat breeders) were included in estrus synchronization protocols, viz.. Mid-cycle PG, Ovsynch and CIDR, and subsequent ovarian follicular/luteal dynamics, blood profile and fertility evaluation. Six subestrus/ repeat breeding postpartum cows each of Gir & Crossbreds (6x2 = 12) having mid cycle mature CL were treated with single i/m injection of 500 ng cloprostenol, and fix timed AI was performed at 72 and 96 hrs post-treatment. Ultrasound scanning of the animals was performed daily from start of treatment till expression of induced estrus/FTAI and/or ovulation with blood sampling. Similarly, six infertile cyclic cows each of Gir and Crossbred category (6x2=12) were treated with standard CIDR protocol and equal numbers (6x2=12) with Ovsynch protocol using FTAI on day 9 and 10. The ovarian changes were monitored daily with blood sampling on day 0 (initiation of treatment), 2, 4, 6, 7 (PG injection), 8,9 (GnRH injection), and day 10 (induced estrus/FTAI). All 36 induced cycling and fix timed inseminated cows together with 24 spontaneously cycling and inseminated cows {cows (n=12) and heifers (n=12), 6 each of Gir and Crossbred type} were further monitored for follicular dynamics daily from the day of AI till day 21 post-AI and then for early pregnancy in non-return cases through uterine scanning with 6.5 MHz transducer, together with jugular blood sampling at weekly interval, i.e. on day 0 (AI), 7, 14, 21 post-AI and then on day 23, 28, 35 and 42 post-AI in non-return cases for assay of blood glucose and plasma total cholesterol, estradiol and progesterone profile. All these inseminated animals (n=60) not returned to estrus again were monitored through transrectal USG (6.5 MHz) for detection of early pregnancy and/or embryonic mortality on day 23, 28, 35 and 42 post-AI, and the findings were correlated with plasma P4 on day 21 and per rectal palpation on day 60 post-AI. The characteristics of dominant and ovulatory follicles observed between two consecutive estruses in 24 unbred animals, comprising of 12 heifers and 12 cows, 6 each of Gir and HF x K crossbred genotype, using 7.5 MHz tranrectal linear array transducer indicated the presence of two- and three-wave cycles in 66.66 (n=12) and 33.33 (n=6) per cent, respectively, of all heifers and crossbred cows, while in Gir cows the frequency of 2- and 3-wave cycles was equal. None of the animals showed single-wave cycle. The second wave appeared earlier in the estrous cycle with 3-wave than with 2-wave (9.00±1.00 vs 10.00±0.58 days; P<0.01). The persistence of the first dominant follicle and duration of regression phase were significantly (P<0.05) longer in 2-wave than in 3-wave cycles (20.25±0.25 vs 19.00±0.00 days and 7.25±0.62 vs 5.00±0.00 days). The maximum diameter of ovulatory follicle was significantly higher (P<0.05) in 3-wave as compared to 2-wave pattern (13.70±0.10 vs 12.55±0.27 mm). The linear growth rate of first dominant follicle was significantly (P<0.01) higher in 2-wave as compared to 3-wave cycle (1.09±0.19 vs 0.46±0.11 mm/day). Similarly, the linear growth of ovulatory follicles was significantly (P<0.05) higher in 3-wave than the 2-wave cycles in all animals. The duration and end day of second dominant follicle were significantly (P<0.05) longer in 2-wave as compared to 3-wave cycle (12.00±0.70 vs 6.00±0.00, 21.50±0.28 vs 14.50±0.50). The emergence of ovulatory follicle was significantly (P<0.01) earlier in 2- wave (9.50±0.50 days) as compared to 3-wave cycle (14.50±0.50 days). Similarly, the duration of ovulatory follicles differed significantly (P<0.01) between 2- and 3-wave cycles (12.00±0.70 vs 7.00±0.00 days). The diameter of ovulatory follicle of 2-wave cycle was significantly (P<0.05) larger than 3~wave cycle (17.22±0.79 vs 14,65±0.45 mm). The ovulatory follicle of 2-wave cycle in crossbred cows appeared significantly (P<0.01) earlier than 3-wave cycle (days 9.00±0.71 vs 14.50±0.50). The persistence of ovulatory follicle was significantly longer (P<0.01) in 2-wave as compared to 3-wave cycle (12.00 ±0.70 vs 7.50±0.50 days). Two- and three-wave cycles differed significantly (P<0.05) with respect to the mean interovulatory interval (22.33±0.33 vs 24.00±0.00 days). In Crossbred cows with 2-wave cycle, there was no relationship between the development of first dominant follicle and presence of CL in either of the ovaries, while in 3-wave cycle the first dominant follicle and the ovulatory follicle developed on opposite side to the ovary bearing the CL, while the second dominant follicle developed on the same ovary bearing the CL in both Gir and Crossbred cows. Similarly, no pattern was observed for the development of ovulatory follicle with respect to first dominant follicle on the ovaries in 2-wave cycle. However, the proportion of ovulatory follicles was higher on opposite side as compared to same side to the first dominant follicle in 2-wave cycle in both the breeds. In 3-wave cycle the second dominant follicle developed opposite to first dominant follicle and the ovulatory follicle developed opposite to second dominant follicle in all the animals. Thus, there was no difference in the pattern of this phenomenon between two breeds studied. In Gir and Crossbred cows, there were positive correlations between CL diameter and plasma P4 values (r = 0.82 & 0.72), and follicle size and E2 values (r = 0.69 «& 0.36). The average plasma P4 values were higher (P<0.01) and E2 values lower on day 7 and 14 than on day 0 and 21 post-estrus, with corresponding larger CL and smaller follicular size in both Gir and Crossbreds. The profiles of follicular growth and regression compared for first 21 days post-AI in conceived and in non-bred cyclic Gir & Crossbred cows revealed the fi-equency of occurrence of two follicular wave up to 75.00 (6/8) per cent in pregnant and 58.33 (7/12) per cent in non-bred cyclic cows, while the remaining seven cows (2 pregnant and 5 cyclic) showed three follicular waves. Almost similar patterns in the follicular growth characteristics, viz., beginning day, end day and duration (days) of first dominant follicle were observed between pregnant and non-bred cyclic cows (0.75±0.29 vs 0.50±0.29, 9.25±0.48 vs 8.25±0.25 and 8.50±0.48 vs 7.75±0.48). However, the linear growth rate (mm/day) of first dominant follicle was significantly lower (P<0.05) in pregnant as compared to non-bred cyclic cows (0.93±0.07 vs 1.21±0.12). The difference was also significant (P<0.01) in the beginning day (15.75±0.25 vs 14.00±0.41) and the maximum diameter of first dominant follicle (11.72±0.22 vs 14.40±0.24 mm) between above two groups. Similarly, the duration (days) of regression phase and linear regression rate (mm/day) of first dominant follicle (4.75±0.65 vs 7.00±0.40 and -1.07±0.07 vs -1.39 vs 0.09) and the maximum diameter of second dominant follicle (11.70±0.22 vs 16.40±1.04 mm) were significantly (P<0.01) lower in pregnant as compared to normal cyclic cows. In all, 60 spontaneous (n=24) or induced cyclic (n=36) cows were inseminated and those did not return to estrus by day 21 after AI were further scanned four times on day 23, 28, 35 and 42 using trans-rectal linear array transducer of 6.5 MHz frequency to detect early pregnancy and embryonic mortality, if any. The sensitivity of USG was cent per cent on all the 4 days, but the specificity was lower on day 23 and 28 (68.00 % each) as compared to day 35 (92.00 %) and 42 (100%). On day 23 and 28, 8 animals were incorrectly diagnosed pregnant, while on day 35, two and zero animals were wrongly diagnosed pregnant. However, the progesterone assay on day 21 post-AI revealed 9 animals being diagnosed incorrectly pregnant. The sensitivity and negative predictive values were cent per cent on all days by both the methods, but specificity and positive predictive values were lower for pregnancy diagnosis using progesterone assay (64.00 % and 79.55 %) as compared to ultrasound scanning results on all 4 days (day 23 & 28, 68.00 to 81.40 %; day 35, 92.00 - 94.59 % and day 42, 100 %). Similarly, the accuracy was relatively higher with ultrasound scanning than the progesterone assay (100 vs 86.44 %). Based on plasma progesterone profile, 35 cows were correctly diagnosed as pregnant (P4, 6.48±0.38 ng/ml) and 16 cows as non-pregnant (P4, 0.45±0.13 ng/ml). However, 9 animals were incorrectly diagnosed as pregnant (P4, 4.14±0.12 ng/ml) and no animal was incorrectly diagnosed as non-pregnant as compared to the results of pregnancy diagnosis on day 60 by rectal palpation. Out of 9 cows, which were incorrectly diagnosed pregnant on the basis of plasma P4 profile on day 21 post-AI, 6 were found pregnant on day 23 and 28, but only three were found pregnant on day 35 by ultrasonography indicating early embryonic mortality in three animals (3/35; 8.57%) between day 28 and 35, The technique of ultrasound scanning facilitated the diagnosis of all non-pregnant animals as early as on day 23 post-service. The results indicated that day 35-42 is the earliest possible time when pregnancy diagnosis should be attempted using ultrasound for maximum accuracy and specificity. The conception rates of 12 subfertile cows, each subjected to CIDR, Ovsynch and Mid-cycle PGF2a treatment protocols were 41.66, 41.66 and 33.33 per cent, respectively, at induced estrus. The corresponding conception rates at second cycle post-treatment were 28,57, 42,85 and 25,00 per cent; and at third cycle 40,00, 50.00 and 33.33 per cent, with the overall conception rates of 3 cycles as 75.00, 83.33 and 66.66 per cent. In untreated cyclic control group, out of 24 animals (12 Gir and 12 Crossbreds) inseminated, the conception rates at first, second and third service and overall of 3 services were 20.83, 21.05, 26.66 and 54.16 per cent, respectively. The results obtained using all 3 treatment protocols were much better than that of untreated cyclic control animals. Gir cattle in fact responded better with CIDR, while Crossbreds showed better results with Ovsynch protocol, and the response with Mid-cycle PGF2a treatment was relatively poor and almost same in both the classes of animals. The follicular dynamics, CL size, plasma progesterone, estradiol-17p, total cholesterol and blood glucose profiles were studied in Mid-cycle PGF2a treated as well as CIDR and Ovsynch treated Gir and Crossbred cows from the day of initiation of treatment until day of induced estrus/FTAI. The pooled mean CL size in cows as monitored by transrectal ultrasonography at 0, 24, 48 and 72 (estrus/AI) hrs of PG treatment was found to be 14.02±0.46, 12.68±0.49, 10.26±0.62 and 7.70±1.16 mm, respectively, and that of follicle 4.42±0.22, 5,37±0.35, 8.52±0.59, 12.94±0.74 mm. The corresponding plasma progesterone concentrations were 5.22±0.43, 3.10±0.36, 1.14±0.32 and 0.48±0.31 ng/ml, respectively, and those of estradiol-17p 19.42±2.07, 24.33±2.33, 41.25±4.40, 51.41±2.02 pg/ml. The values of CL size and plasma P4 were significantly (P<0.01) higher and those of follicle size and E2 lower on the day of PG treatment indicating the presence of mature functional CL on the ovary. The CL size reduced and plasma progesterone concentration dropped significantly with parallel increase in follicle size and plasma E2 within 48-hrs of PG treatment. The values of CL size and plasma P4 were the lowest or at basal level and those of follicle and E2 at peak on the day of induced estrus/AI, around 72-hrs posttreatment. No significant variation was noted in any of the traits between breeds or at any of the intervals studied within the breed, except at 72-hrs of PG treatment wherein the values of CL size and plasma P4 were significantly (P<0.05) higher in crossbreds as compared to Gir, suggesting breed difference in the action of PG and duration of induced luteolysis. The conceiving and non-conceiving cows revealed identical pattern and values of CL and follicle size as well as plasma P4 and E2 concentrations till 48-hrs of mid-cycle PG injection, but by 72-hrs of treatment the drop in CL size and plasma P4 were drastic and significant over 48-hrs values in conceiving cows than the non-conceiving cows. The pooled mean concentration of blood glucose recorded at 0, 24, 48, 72 hrs after PG treatment was 56.78±2.25, 57.34±2.00, 58,76±2.06, 60.78±1,61 mg/dl, respectively. The corresponding plasma total cholesterol concentrations were 166.84±3.73, 166.36± 2.33, 173.42±2.65, 169.19±3.89 mg/dl, respectively. The values of none of these traits varied significant between different intervals post-PG-treatment, and the values were more or less similar in both the breeds, and no significant variation was noted in blood glucose or plasma cholesterol profile at any of the intervals studied between breeds. The follicular dynamics, CL size, plasma progesterone, estradiol-17p, cholesterol and blood glucose profiles were also studied in Gir and Crossbred cows on day 0 (Wg CIDR insertion), 2, 4, 6, 7 (CIDR removal & PG injection), 8 and 9 (GnRH injection) of CIDR (1.38 g hydroxyl-progesterone in silastic coil) treatment, and on day 10 (induced estrus/FTAI). The pooled mean CL size in cows on these was found to be 11.09±0.71, 10.97±0.69, 10.80±0.50, 10.60±0.43, 10.63±0.49, 8.25±0.18, 5.65±0.38 and 5.21±0.67 mm, respectively and that of follicle size 7.70±0.59, 7.23±0.36, 8.68±0.71, 8.63±0.63, 9.02±0.67, 10.50±0.61 12.48±0.72, 7.50±1.00 mm, respectively. The corresponding plasma P4 concentrations were 4,55±0.34, 5.54±0.49, 5.12±0.31, 4.73±0.24, 4.66±0.36, 1.78±0.21, 0.67±0.12, 0.13±0.02 ng/ml, respectively, and those of estradiol-17p 14.16± 1.94, 25.50±2.40, 29.16±2.94, 37.16±3.39, 30.75±1.92, 40.75±2.75, 49.66±4.03, 33.50 ±7.02 pg/ml. The values of CL size and plasma P4 were significantly (P<0.01) higher and follicle size and E2 lower on the day of CIDR insertion indicating that the animals were cyclic with presence of mature functional CL on the ovary when the treatment was initiated. The CL/ follicle size and plasma progesterone/estradiol concentrations were maintained for 7 days until CIDR was removed and PG was injected i/m, which resulted in luteolysis with sudden and significant drop in CL size and plasma progesterone within 24- hrs with further drop to basal level with concurrent rise in follicle size and plasma E2 levels by next 48-hrs, i.e. the day of induced estrus/FTAI due to withdrawal of negative feedback effect of plasma P4 on the pituitary and hypothalamus, thus inducing ovulatory estrus within 72-hrs. No significant variation was noted in any of these traits at any of the intervals studied, between or within Gir and Crossbred cows. The values of blood glucose and cholesterol were more or less similar in both the breeds, as were found in Mid-cycle PG treated group, and no significant variation was noted at any of the intervals post-CIDR treatment between breeds or within breed. Although the blood glucose levels were apparently higher and cholesterol lower in Gir cows as compared to Crossbreds during the CIDR treatment phase until induced estrus. The effect of Ovsynch treatment on the follicular dynamics, CL size, plasma progesterone, estradiol-17p, total cholesterol and blood glucose profiles studied in infertile cyclic Gir and Crossbred breed cows on the same days as with CIDR protocol was almost similar with identical pattern and values to those of CIDR treatment group. All these parameters were also studied in normal cyclic control post-pubertal heifers (6) and postpartum cows (6) of Gir and Crossbred breeds each at weekly interval, together with those cows induced to estrus and inseminated at fixed time under three synchronization protocols, from day 0 (estrus/AI) to day 21 post-AI and then on day 23, 28, 35 and 42 in non-return cases. One cows in each synchronization treatment underwent embryonic mortality between day 28 and 35 post-AI as confirmed by transrectal USG. The pooled mean CL size in 12 heifers as monitored by ultrasonography on day 0 (estrus/AI), 7, 14, 21, 23, 28, 35 and 42 post-AI was found to be 4.72±0.33, 12.30±0.55, 12.72±0.62, 8.74±1.39, 9.47±1.17, 11.83±0.87, 14.44±0.61 and 14.50±0.67 mm, respectively, and that . of follicle size 12.70±0.51, 5.86±0.52, 5.23±0.45, 9.21±0.96, 7.4U0.91, 6.14±0.48, 4.80±0.51 and 4.94±0.30 mm, respectively. The corresponding plasma progesterone concentrations were 0.49±0.10, 3.16±0.21, 5.18±0.41, 3.01±0.99, 3.50±0.96, 5.66±0.73, 8.54±0.21 and 8.96±0.15 ng/ml, respectively, and those of estradiol-17p were 56.08±2.98, 29.58±2.52, 20.25±2.43, 38.08±5.70, 22.66±4.97, 21.58±2.84, 15.80±2.53 and 22.40±3.29 pg/ml, respectively. Very similar values and trend of observations were recorded for CL and follicular size as well as plasma progesterone and estradiol-17p profile in postpartum cows also from the day of estrus/breeding till day 42 post-AI. The values of CL size and plasma P4 were the lowest or at basal level on the day of estrus/AI, increased highly significantly (P < 0.01) by day 7 and 14, dropped down around day 21-23 and again rose significantly at day 35 and 42 post-AI, with concurrent inverse trend in follicle size and plasma E2 profile. These trends were associated with establishment of pregnancy and return of rest of the animals to next cycle around day 21- 23. No significant variation was noted in CL or follicular size and plasma progesterone or estradiol-17P profile at any of the intervals studied and the trend was the same in both the Gir and Crossbred heifers as well as cows. Further, the conceiving and non-conceiving animals of Gir and Crossbred breed (irrespective of parity) revealed identical pattern and values of CL/follicle size and plasma P4, E2 concentrations till day 14 post-AI, and there was significant drop in CL size as well as progesterone profile in non-pregnant animals with increase in follicle size and plasma E2 profile due to luteolysis, folliculogenesis and res-establishment of next cycle around day 21-23; and persistence of CL and continuance of progesterone production in conceived ones. The pooled mean concentration of blood glucose in heifers on day 0 (estrus/AI), 7, 14, 21, 23, 28, 35 and 42 post-AI was found to be 64.35±3.21, 68.71±2.54, 66.41±2.63, 68.67±2.10, 66.91±2.08, 64.43±2.30, 60.96±1.69 and 61.18±1.58 mg/dl, respectively. The corresponding cholesterol levels were 172.73±5.27, 175.91±3.69, 181.01±1.85, 176.90± 3,33, 173.90±2.65, 174.81±2.19, 170.50±2.60 and 169.79±1.43 mg/dl, respectively. Very similar values and trend of observations were recorded for blood glucose and plasma total cholesterol profile in postpartum cows from the day of estrus/breeding till day 42 post-AI. The values of none of these traits varied significantly between intervals post-estrus/AI, No significant variation was noted between Gir and Crossbred heifers as well as cows in blood glucose or plasma cholesterol profile at any of the intervals studied and the trend was the same. Further, the conceiving and non-conceiving animals of Gir and Crossbred breed (irrespective of parity) revealed identical pattern and values of blood glucose and plasma cholesterol concentrations till day 42 post-AI. Very similar trend and values of follicular dynamics, CL size, plasma P4, E2, total cholesterol and blood glucose were also found in all three groups of estrus synchronized cows of Mid-cycle PG, CIDR and Ovsynch protocols (12 each) at weekly interval from day 0 (induces estrus/FTAI) to day 21 post-AI and then on day 23, 28, 35 and 42 in nonreturn cases, hence the data are not repeated again for these groups. It is concluded that the ultrasound scanning is a very usefiil technique to study follicular dynamics as well as to detect early pregnancy and embryonic mortality in bovines. The Gir and Crossbred cows evince either 2- or 3-follicular waves per cycle, but not the single wave cycle. Ultrasonographic technique is helpful in detection of nonpregnancy as early as day 23 post-service with cent percent negative predictive value. There is regular growth and development of dominant follicles during early stages of pregnancy as a safe guard in the event of early embryonic mortality to evince next cycle in time. Plasma progesterone profile on day 21 post-breeding is also a very good index for detection of pregnancy with 79.55 per cent positive predictive value and cent per cent negative predictive value. The CL diameter and plasma P4 values as well as follicle size and E2 values were positively correlated. The results in general did not reveal any specific role of glucose or cholesterol in reproductive processes. Of the different synchronized treatment protocols, the highest overall conception rate was achieved with Ovsynch (83.33%) followed by CIDR (75.00%) and Mid-cycle PGF2a (66.66%) treatment and the least in control group (54.16%), hence the Ovsynch and CIDR treatments can be recommended to the field veterinarians for improving the fertility of infertile cattle.