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ThesisItem Open Access Fertility Evaluation of Chilled Buck Semen using different Extenders(SKUAST Kashmir, 2022) Nahida Yousuf; Arjuma KhatunSemen was collected from selected four healthy cross bred Boer bucks by using sterile artificial vagina maintained at Mountain Research Centre for Sheep and Goats, SKUAST-K Shuhama Kashmir. The current study was conducted in three experiments viz. 1) Effect of washing on preservation of buck semen at 4ºC during different time intervals. 2) Comparing egg yolk based extender containing 2.5% egg yolk and commercial soybean-lecithin based extender i.e. Ovixcell on preservation of buck semen at 4ºC during different time intervals. 3) Artificial insemination following estrus synchronization with chilled buck semen extended with egg yolk and soybean lecithin-based extender. Semen samples having sperm motility greater than 70% were pooled and divided into two aliquots in all the three experiments. In first experiment two aliquots were designated as T1 and T2 based on the presence or absence of seminal plasma, then both the aliquots were extended with TCFEY containing 2.5% egg yolk and stored at 4°C upto 72 h. Similarly in experiment-II two aliquots were designated as T3 and T4, extended with 2.5% egg yolk based and commercial soyabean lecithin based extender, respectively. In experiments I and experiment II, the quality of spermatozoa was evaluated on the basis of percent sperm motility, live sperm percentage, morphological sperm abnormalities, hypo osmotic swelling test (HOST), intact acrosome and oxidative stress measured in terms of malondialdehyde (MDA) production in sperm cells, observed after extension in particular extenders at 0 h, 24 h, 48 h and 72 h, preserved at 4°C. In addition to this MDA production in seminal plasma was also measured in experiment II. In experiment III, a total of 45 number cross bred Boer does aged 1.5- 8 years were selected and divided into 5 groups designated as natural service group (GI) (N=10) as CON, fresh egg yolk (GII) (N=10), 72 h preserved egg yolk (GIII) (N=8), fresh Ovixcell (GIV) (N=9) and 72 h preserved Ovixcell (GV) (N=8). In all the five groups, intravaginal progesterone sponges were kept for 12 days and prostaglandin shot was given 24 h prior to sponge withdrawal. Further, except CON group GnRH was given 36 h post sponge removal. In CON group, one breeding buck was kept with the does for one cycle (20 days). In remaining four groups fixed timed double AI was done at 48 and 60 hours using 0 h fresh semen extended with TCFEY (GII) and ovixcell (GIV) and 72 h preserved semen extended with same extenders designated group GIII and GV respectively. Fertility parameters recorded in experiment III were pregnancy rate, kidding rate and prolificacy rate. Also, blood sampling was done at day 0 (day of AI), day 12, day 21 and day 40 post AI to determine progesterone concentration. In experiment I, the percent sperm motility was significantly (P<0.05) higher in T1 than T2 treatment group up to 72 h preservation at 4°C. The live sperm count was significantly (P<0.05) higher at 72 h in T1 than T2 treatment group. The HOST reacted spermatozoa was significantly (P<0.05) higher at 0 h and 24 h and intact acrosome at 0 h and 72 h at 4°C in T1 than T2 treatment group. In experiment-II, the percent sperm motility was significantly (P<0.05) higher in T3 than T4 treatment group during 72 h preservation at 4°C. The live sperm percentage was significantly (P<0.05) higher at 72 h, HOST reacted spermatozoa at 24 h in T3 than T4 treatment group. MDA level in seminal plasma was significantly (P<0.05) lower at 72 h in T3 than T4 treatment group. Spermatic MDA level was significantly (P<0.05) higher at 24 h and 48 h in T4 than T3 treatment group during preservation at 4°C. In experiment-III, the pregnancy rate and kidding rate were significantly (p<0.05) higher for GI (CON) than GIV and GV The serum progesterone concentration was significantly(P<0.05) higher in pregnant than non-pregnant does at day 12 (13.93±1.77), at day 21 (19.80±1.67) and at day 40 (23.20±1.18). Further, within pregnant does the mean serum progesterone concentration increased significantly(P<0.05) from day 0 to day 40. However, within non pregnant does, the values decreased significantly(P<0.05) from day 0 to day 40. In conclusion, lower percent of egg yolk (2.5%) in Tris based extender may provide better buck semen quality without washing at 4°C for 72 hours and is comparatively better than soya lecithin-based extender (ovixcell). Also chilled (4°C) buck semen extended with 2.5% egg yolk and Ovixcell, can be used for artificial insemination preserved up to 72 hours and 48 hours, respectively.ThesisItem Open Access Effect of Equilibration Period, Vitrification Technique and Antioxidants on the Post-thaw Quality of Vitrified Ovine Ovarian Cortical Tissue(SKUAST Kashmir, 2022) Masrat un Nisa; Malik, Asloob AhmadThe present study involved three experiments to achieve the following objectives. (i) To determine the effect of equilibrium time periods on post-thaw viability, morphology of retrieved oocytes and histology of vitrified ovarian tissue, (ii) To investigate the effect of two vitrification techniques i.e., Cryovial with vitrification solution (CV), Direct cover vitrification (DCV) on post-thaw viability, morphology of retrieved oocytes and histology of vitrified ovarian tissue, (iii) To examine the effect of different antioxidants i.e., Resveratrol, Zinc sulphate, Curcumin & Quercetin on post-thaw viability, morphology of retrieved oocytes and histology of vitrified ovarian tissue. In experiment 1, after processing and preparation of ovarian tissue fragments, the fragments were exposed to two equilibration timings 25/15 min (Protocol-I) and 10/5 min (Protocol-II) followed by vitrification. Out of 145 and 102 immature oocytes retrieved from ovarian cortex, 130 (89.6%) and 90 (88.2%) oocytes were viable, respectively in Protocol I and Protocol II. The number of oocytes found morphologically normal were 119 (82%) and 80 (78.4%), respectively in Protocol I and Protocol II and the values differed non-significantly (P>0.05) between the two protocols. Histological assessment demonstrated that the percentage of intact follicles was significantly (P≤0.05) high (91.97%±2.89) in fresh control group compared to both Protocol-I (26.67%±5.49) and Protocol-II (48.91%±5.80). However, the percentage of intact follicles was significantly (P≤0.05) better in Protocol-II (48.91%±5.80) compared to Protocol-I (26.67%±5.49). In experiment 2, vitrification techniques CV and DCV were compared for the viability and morphology of retrieved oocytes and histology of vitrified ovarian tissue. In CV technique, the tissue fragments were loaded into cryovials pre filled with 1 ml vitrification solution-II, followed by plunging into LN2 canister. In DCV technique the ovarian tissue fragments were loaded into empty cryovials and immersed into LN2 canister. In DCV and CV techniques, a total of 178 and 190 immature oocytes were retrieved from ovarian cortex. The number of viable oocytes following CV technique were significantly (P≤0.05) higher than DCV method {164 (86.3%) vs 129 (72.4%)}. The number of oocytes found morphologically normal were 132 (74.1%) and 135 (71%), after following DCV and CV techniques respectively and the values did not differ significantly (P>0.05). Histological assessment of the ovarian cortical tissue showed that the percentage of intact follicles in fresh control was significantly (P≤0.05) high in fresh control compared to those following DCV (28.46±8.09) and CV (47.61±12.39) techniques. However, the percentage of normal intact follicles was non-significantly higher for CV (47.61±12.39) than in DCV technique (28.46±8.09). In experiment 3, four different antioxidants i.e., Resveratrol (20 μM), ZnSO4 (500 μM), Curcumin (25 μM) and Quercetin (1 μM) were evaluated after their addition to the vitrification and warming media for the viability and morphology of retrieved oocytes and histology of vitrified ovarian tissue The number of oocytes retrieved from ovarian tissue from the above mentioned antioxidants and vitrified control were 34, 41, 26, 31 and 46 respectively. Among these number of viable oocytes were 24 (70.5%), 30 (73.1 %), 20 (76.9%), 26 (83.8%) and 33 (71.1%) and the number of oocytes found morphologically normal were 24 (70.5%), 26 (63.4%), 18 (69.2%), 21 (67.7%) and 34 (73.9%) for above mentioned different antioxidants and vitrified control respectively. Non-significant (P>0.05) differences were found between different treatment groups. Histomorphological evaluation of the ovarian cortical tissue showed that the percentage of intact follicles was significantly (P≤0.05) high in fresh control (84.19±3.89) than in other groups. Non-significant difference was found between Resveratrol (50.2±5.5), Curcumin (48.71±5.74), Quercetin (51.59±4.77) and Vitrified control (42.73±6.13) groups however the ZnSO4 supplemented group (23.1±8.54) differed significantly (P≤0.05) from other antioxidant groups but was non-significant (P>0.05) with vitrified control group (42.73±6.13). Thus from the present study it was concluded that: (i) short time exposure i.e., 10/5 (Protocol-II) preserved the follicular morphology significantly better than long time exposure i.e., 25/15 (Protocol-I). (ii) CV technique was non-significantly efficient for morphological integrity for sheep ovarian tissue vitrification than DCV. (iii) Addition of Antioxidants i.e., Resveratrol, Curcumin and Quercetin non-significantly improved the follicular integrity after vitrification.ThesisItem Open Access Effect of supplementation of honey and antioxidants on the quality of cryopreserved ram semen(SKUAST Kashmir, 2016) Banday, Mohamad Naiem; Lone, Farooz AhmedSix adult healthy cross bred rams maintained at Mountain Research Centre for Sheep and Goats, SKUAST-K Shuhama Kashmir were selected for collection of semen. The semen from selected rams was collected by artificial vagina method. The present study was conducted in two experiments viz. 1) Effect of addition of honey to tris extender on the quality of ram semen at post thaw. 2) Effect of addition of antioxidants namely Taurine, Quercetin and Reduced glutathione to tris extender on the quality of ram semen at post thaw. Semen samples showing sperm motility greater than 70% were pooled and divided into four aliquots in both the experiments.The four aliquots were designated according to the treatment groups-CON, T1, T2 and T3 and were extended with respective tris extender based on presence or absence of antibiotics and variation in concentration of honey in experiment-1 and containing different antioxidants in experiment-2. Prior to freezing a portion of semen sample was taken and maintained at 30-34oC for pre freeze semen quality evaluation. In experiment-1, the four treatment groups consisted of CON (Tris extender), T1 (Tris+2.5% Honey, without antibiotics and fructose), T2 (Tris+5% Honey, without antibiotics and fructose) and T3 (Tris+7% Honey, without antibiotics and fructose). Similarly in experiment-2, the four treatment groups consisted of CON (Tris extender), T1 (Tris+40 mM Taurine), T2 (Tris+0.000014 mM Quercetin) and T3 (Tris+5 mM reduced glutathione). The quality of spermatozoa was determined in both the experiments on the basis of percentage of sperm motility, live sperm count, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa, both at pre freeze and post thaw. In addition to this, total viable count was also determined at post thaw in experiment-1 and oxidative stress measured in terms of malondialdehyde (MDA) production both in seminal plasma and sperm cell was also determined at post thaw in experiment-2. In experiment-1, the percent sperm motility was same for all the control and treatment groups at pre freeze. However, at post thaw, T1 maintained non significantly lower (p>0.05) sperm motility than CON but significantly higher (p<0.05) sperm motility than T2 and T3. The percent live sperm count, intact acrosome and HOST reacted spermatozoa were significantly higher (p<0.05) for CON than all other treatment groups at post thaw. Among treatment groups, T1 maintained significantly higher (p<0.05) percentage of live sperm count, intact acrosome and HOST reacted spermatozoa than T2 and T3. The total viable count in terms of colony forming units/ml at post thaw was significantly lower (p<0.05) for control than all the treatment groups. In experiment-2, T1 maintained significantly higher (p<0.05) post thaw sperm motility and live sperm count than CON and T3. The percent intact acrosome did not differ significantly among CON, T1 and T2. However, T3 maintained significantly higher (p<0.05) number of spermatozoa with intact acrosomes. The HOST reacted spermatozoa was significantly higher (p<0.05) for T1 than CON and other treatment groups (T2 & T3). Seminal plasma MDA level (nmol/ml) in T1 was significantly lower (p<0.05) than CON and T2 and non significantly lower than T3 at post thaw. However, spermatic MDA levels differed non significantly among the control and treatment groups. In conclusion, addition of honey cannot replace the use of antibiotics in semen extenders to take care of heavy microbial load, but may replace fructose because the sperm quality was at par with the control when added at 2.5% to tris extender. Levels above 2.5% resulted in severe deterioration of sperm quality, thus honey upto levels of 2.5% may be added to the basic tris extender for cryopreservation of ram semen. Taurine at the rate of 40mM should be used as an antioxidant supplement in tris extender for better quality of ram semen at post thaw.ThesisItem Open Access Ultrasound guided diagnosis and therapeutic management of delayed ovulating and anovulating repeat breeding cows(SKUAST Kashmir, 2018) Mir, Naseer Ahmad; Naikoo, MehrajuddinThe present study was designed with the objectives to diagnose delayed ovulating and/ anovulating repeat breeding cows by palpation per rectum and with the aid of real time transrectal ultrasonography, to evaluate and compare the conception rate following use of hormones (GnRH and hCG) and single and double insemination/s in repeat breeding cows, to detect early pregnancy/ non-pregnancy by transrectal USG on day 26 post-AI and to study the blood serum biochemical parameters in normal cyclic and repeat breeding cows. Crossbred dairy cows from local areas with history of 3 or more than three unsuccessful matings/ inseminations, free from uterine infection and gross ovarian and uterine pathologies were subjected to transrectal ultrasonography for three consecutive days (day 0, day 1st and day 2nd) to asses the status of ovulation/ anovulation. The ultrasound scanning of the cows for confirmation of delayed ovulation and /or anovulation, was conducted using a transrectal/ endorectal probe (5.0 to 7.5 MHz) of the equipment “Esaote My Lab 40- Vet”. Out of total 80 repeat breeding animals which were examined per-rectally and with the aid of trans-rectal USG, 24 animals were found to be having ovulatory defect either delayed or anovulation (30%). They were further differentiated into 22 cows with delayed ovulation and 02 cows with anovulation on the basis of presence or absence of CL during mid cycle on the ipsilateral ovary, respectively. All the 24 ovulatory defective (delayed and anovulation) animals were allotted to four different groups (A, B, C and D) each consisting of 06 animals. An additional group (E) consisting of 06 normally cyclic animals was kept as normal cyclic control. Animals of group A and B were treated with hCG (1500IU I/M) and GnRH (10mcg I/M), respectively. Groups C and D were subjected to double and single insemination, respectively. The group D animals were considered as repeat breeding control. Under group E, healthy normally cyclic cows without history of RB brought to the VCC, FVSc & AH, SKUAST-K, for routine AI were kept as cyclic control animals. The serum samples harvested on day 0 and day 1st of estrus were subjected to estimation of progesterone by ELISA and total serum Cholesterol, Calcium and Phosphorus by using standard kits. The early pregnancy/ non pregnancy was confirmed by USG on day 26 post AI, followed by per rectal examination on day 60 post AI. The first service conception rate of 50.00%, 66.66%, 83.33%, 16.66% and 83.33% were found in Group A, B, C, D and E, respectively. In general, the mean blood serum calcium and phosphorus was found to be significantly higher (p≤0.05) in normally cyclic animals as compared to repeat breeding cows. There were no significant differences in concentration of calcium and phosphorus between day 0 and day 1. The total serum concentration of cholesterol was significantly higher (p≤0.05) in normal cyclic as compared to repeat breeding cows and no significant difference was found between day 0 and day 1 of estrus. Significant (p≤0.05) variations of plasma progesterone level were observed between normal cyclic and repeat breeder cow, and without any significant difference between day 0 and day 1 of estrus, except for the group B (GnRH administered group). In conclusion, trans-rectal USG serves as an excellent tool in diagnosis and confirmation of conditions like delayed and anovulation, and can be used with high 100% specificity for early pregnancy diagnose in bovines as early as 26 days post insemination. Protocols like Double inseminations and hormonal therapies (GnRH and hCH) can be effectively used for improving conception in delayed ovulating and anovulating animals. However double insemination was found most effective. Disturbance of macro-minerals minerals (Ca, P) may affect the reproductive performance in dairy cows, hence, proper balance is a must.ThesisItem Open Access Diagnostic and Therapeutic Evaluation of Subclinical Endometritis in Dairy Cattle(SKUAST Kashmir, 2018) Arshaq Asfar; Khursheed Ahmad SofiThe present study was conducted in 39 crossbred cows (>60 DIM) to evaluate various diagnostic tests and therapeutic regimens for subclinical endometritis (SCE). The diagnostic tests included were leukocyte esterase (LE) strip test, protein strip test, pH strip test, Whiteside test (WST) of EM, bacterial and fungal culture of EM in comparison to endometrial cytology (EC) using cytobrush (CB) as reference diagnostic method. EM was collected aseptically from body of uterus at 8-12 hours after start of estrus. EC was done in 39 cows with ≥4% PMNs as cut-off value for SCE. LE strip test was done by contacting EM with multi-reagent urinalysis reagent strips and results recorded (according to manufacturer recommendations) after 10 minutes. Similarly, protein and pH strip tests were conducted using the same strips and results recorded after 60 seconds. WST of EM was conducted simultaneously using EM. EM from 12 cows positive for SCE by EC was subjected to bacterial and fungal culture. Physical characteristics of EM were also recorded. LE, protein and pH strip tests were evaluated and analyzed statistically using Receiver Operator Characteristic (ROC). To evaluate various therapeutic regimens, 30 animals positive for SCE by EC were divided into five groups of six animals each. Therapeutic regimens include 0.3% Lugol’s iodine (30 ml) I/U for 3 days (G-I), Lenovo AP (30 ml) I/U for 3 days (G-II), Meriflox @ 4-5 mg/kg I/M for 3 days (G-III), Meriflox I/M @ 4-5 mg/kg I/M + AI (G-IV) and no treatment + AI as control (G-V). In G-I, II, and III, WST of EM and AI was done on next estrus to evaluate recovery rate and conception rate. However, in G-IV and V, AI was done on the same estrus to evaluate conception rate. Treatment efficacy was analyzed by recovery rate in the form of negative WST at next estrus (G-I, II, III) and conception rate based on pregnancy diagnosis after two months of AI in all the groups. LE strip test at cut-off value ≥ ± obtained from ROC analysis showed sensitivity (Se) of 86.67% and specificity (Sp) of 66.67%. Similarly, protein and pH strip tests showed Se of 53.33% & 66.67% and Sp of 66.67% & 77.78% respectively. Strip tests were also combined in three combinations viz. LE + protein, LE + pH and LE + protein + pH but no increase in performance was seen but decrease in Se was seen in all the three combination (46.7%, 60% and 33.3% respectively). WST had Se of 63.3% and Sp of 55.6% which was lower compared to LE test. Bacterial growth was seen in 66.7% (8/12) while fungal growth was seen in 41.7% (5/12) animals. Conception rate was affected by physical characteristics as thick consistency, copious volume, hanging stringiness, typical fern pattern and spinnbarkeit value >8 cm showed higher conception rate compared to thin consistency, scanty volume, breaking stringiness, atypical fern pattern and SBK value ≤8 cm respectively. Group-I, II and III showed recovery rate of 83.3% (5/6), 50% (3/6) and 100% (6/6) based on WST and conception rate of 50% (3/6), 50% (3/6) and 66.7% (4/6) respectively. However, no difference was found in the conception rate of G-IV (33.3%) and G-V i.e. control (33.3%) in the present study. Further, treatment cost per animal was Rs 9 for Lugol’s iodine, Rs 135 for Lenovo AP and Rs 230 for Meriflox. In conclusion, LE test can be used as an easy cow side field level test with acceptable Se and Sp than WST in comparison to EC by CB for SCE in cattle. However, no improvement in performance of LE strip test was observed on inclusion of pH and Protein strip tests with LE test. Regarding therapeutic management, Meriflox with AI at next estrus proved to be the best treatment option for SCE but Lugol’s iodine can be used as an economically acceptable treatment option for SCE.ThesisItem Open Access Fertility of Ewes Following Hormone Based Estrus Synchronization and Chemical Induced Cervical Ripening(SKUAST Kashmir, 2016) Fabiha Rasool; Lone, Farooz AhmedThe present study was undertaken in two experiments corresponding to two objectives: a) To compare three different estrous synchronization protocols with respect to fertility in Corriedale sheep. b) To record the fertility in artificially inseminated cross-bred ewes following the intravaginal application of misoprostol and isosorbide mononitrate. In experiment-1, a total of thirty Corriedale ewes of 7-9 years of age were equally divided into three groups designated as SPG, SP and SE. Ewes in all the three groups received intravaginal progesterone sponges for 9 days. Further, ewes in group SP and SPG received PGF2α 24 hours before removal of sponges and in group SPG, GnRH was given on day 12 post tupping. Three healthy breeding rams were kept with the ewes for 20 days. In experiment-2, a total of 24 crossbred ewes were divided randomly into three groups designated into three groups, designated as CON (control), MIS (misoprostol) and IMN (isosorbide mononitrate). Estrus in all the ewes was induced by placing intravaginal progesterone sponges for 11 days along with an injection of PGF2α 24 hours before sponge removal. Drug delivery devices loaded with misoprostol and isosorbide mononitrate were placed intravaginally in group MIS and IMN at 12 hours before first AI. Fixed time AI was done twice at 48 and 60 hours after sponge removal using 12-24 hour old chilled semen. Fertility parameters recorded for experiment-1 were time to onset of estrus, estrus response rate, pregnancy rate, lambing rate and prolificacy rate whereas for experiment-2, the fertility parameters recorded were pregnancy rate, lambing rate and prolificacy rate. Blood sampling was done at day 0, at 48 hours of sponge removal, day 15 post mating and day 35 post mating in experiment-1 whereas the corresponding stages in experiment -2 were day 0, day of AI, day 15 post AI and day 35 post AI. The serum harvested was analyzed for nitric oxide, ascorbic acid and progesterone concentration. In experiment-1, time to onset of estrus was more compact in group SE and SPG, however overall estrus response rate was similar in all the three groups. The pregnancy and lambing rates were significantly higher (P<0.05) in group SP and SPG as compared to group SE. Serum nitric oxide and ascorbic acid levels were significantly (p<0.05) higher in pregnant ewes at day 35 post mating. The serum progesterone level in both pregnant and non-pregnant ewes showed a significant decline (P<0.05) from day 0 to day 11. However, the levels were significantly higher (P<0.05) in pregnant ewes as compared to non-pregnant ewes from day 15 to day 35. In experiment-2, the pregnancy and lambing rates were non-significantly (P<0.05) higher in group IMN (62.5%) as compared to group MIS (25%) and CON (25%). The serum nitric oxide level in pregnant ewes was significantly higher (P<0.05) at day 35. The serum ascorbic acid level was significantly higher in pregnant ewes from day 15 to day 35 as compared to non-pregnant ewes. The serum progesterone level was significantly higher (P<0.05) in pregnant ewes at day 35 as compared to non-pregnant ewes. The progesterone level declined significantly (P<0.05) from day 0 to day of AI, followed by a non-significant increase upto day 35. In conclusion, application of intravaginal progesterone sponges for 9 days followed by PGF2α at 24 hours before sponge removal (SP protocol) yielded maximum fertility rate in naturally bred aged Corriedale ewes and artificial insemination in estrus induced cross bred ewes together with the use of NO donor (IMN) intravaginally 12 hours before AI produced higher conception rate.ThesisItem Open Access Protective effect of different antioxidants on ram epididymal spermatozoa exposed to pro-oxidant(SKUAST Kashmir, 2017) Muzamil Rashid; Naikoo, MehrajuddinA total of forty eight (48) testicles from slaughtered adult healthy rams of 2-3 years of age were collected from the two slaughter houses of Srinagar district. The testicles were processed and incision method was applied to recover the spermatozoa from the cauda epididymis. The present study was conducted in two experiments aimed at 1). To evaluate and compare the adverse effects of two different pro-oxidants on ram epididymal spermatozoa. 2). To evaluate and compare the protective effects of different antioxidants on ram epididymal spermatozoa exposed to pro-oxidant. Sperm samples showing sperm motility greater than 70% were pooled and divided into three (3) aliquots in former experiment and divided into five (5) aliquots in the later experiment. The three aliquots were designated according to the treatment groups- control (CON), treatment 1 (T1) and treatment 2(T2) and were supplemented based on presence or absence of pro-oxidants in experiment-1 and five aliquots containing different antioxidants in experiment-2 were designated as CON, T1, T2, T3 and T4. Prior to incubation at 37o C for six (6) hours, a portion of sperm sample was taken and maintained at 30-34oC for pre-incubation sperm quality evaluation. In experiment-1, the three treatment groups consisted of CON (sperm sample), T1 (sperm sample +10 µM sodium arsenite) and T2 (sperm sample +150 µM ferrous sulphate + 750 µM ascorbic acid). Similarly in experiment-2, the four treatment groups consisted of CON (sperm sample + 150 µM ferrous sulphate + 750 µM ascorbic acid), T1 (sperm sample +150 µM ferrous sulphate + 750 µM ascorbic acid + 40 mM Taurine), T2 (sperm sample +150 µM ferrous sulphate + 750 µM ascorbic acid + 50 µM Resveratrol), T3 (sperm sample + 40 mM Taurine) and T4 (sperm sample + 50 µM Resveratrol). The quality of spermatozoa was determined in both the experiments on the basis of percentage of sperm motility, live sperm count, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa, at time 0 hour, 2 hour and 6 hour post-incubation. In addition to this, oxidative stress measured in terms of malondialdehyde (MDA) production in sperm cell was also determined at 0 hour, 2 hour and 6 hour in both experiment-1 and experiment-2. In experiment-1, the percent sperm motility was same for all the control and treatment groups at 0 hour. However, at 2 hour and 6 hour, T2 maintained significantly lower (p 0.05) than T1. The percent live sperm count, intact acrosome and HOST reacted spermatozoa were significantly higher (p0.05) percentage of live sperm count, intact acrosome and HOST reacted spermatozoa than T1 but significantly lower (p 0.05) from T1. The administration of resveratrol (RES) and taurine (TAU) to the FeAA untreated groups led to a non-significant improvement of per cent live sperm (p > 0.05) in case of T4 in relation to T3. The spermatic MDA levels differed non-significantly among the control and treatment groups. In conclusion, the present study provides evidence that sodium arsenite and ferrous ascorbate show a negative influence on sperm structural integrity and functional activity under in vitro condition and hence, may be used routinely in the in vitro studies to induce oxidative stress. RES and TAU were able to compensate the adverse effect on spermatozoa vitality, functional activity and antioxidant capacity as a consequence of ferrous ascorbate-associated oxidative damage and preserve the fertilization potential of male reproductive cells.ThesisItem Open Access Preservation of ram semen at 4°C using different extenders and antioxidants(SKUAST Kashmir, 2015) Rather, Haneef Ahmad; Rafiqul IslamEjaculates were collected from six adult healthy rams maintained at Mountain Research Centre for Sheep and Goats, SKUAST-K, Shuhama Kashmir. The present study was conducted in two phases viz. Phase Ι: Effect of different extenders viz. Tris citric acid fructose egg yolk (TCFEY), Tris citric acid glucose egg yolk (TCGEY), Egg yolk citrate fructose (EYCF) and Egg yolk citrate glucose (EYCG) on the quality of ram spermatozoa during preservation at 4°C and Phase ΙΙ: Preservation of ram semen at 4°C using antioxidants like ascorbic acid (4.5mg/ml), butylated hydroxytoulene (BHT, 1 mM) and taurine (25 mM). The extender which showed best results in Phase I was used in phase ΙΙ as extender using different antioxidants. Semen samples showing more than 3+ mass motility and 70% progressive motility were pooled and subsequently divided into four aliquots in both phases. In phase I, each aliquot was extended separately in four different extenders viz. TCFEY, TCGEY, EYCF and EYCG and stored at 4°C up to 72h. The quality of spermatozoa on the basis of percentage of sperm motility, live sperm, morphological abnormalities, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa was evaluated immediately after extension in particular extenders (0 h), 24 h, 48 h and 72 h after preservation at 4°C. In phase Ι, the percent sperm motility was significantly (P0.05) amongst the extenders during the preservation of spermatozoa. The morphological abnormalities were significantly (P0.05) amongst the extenders containing different antioxidants and the control. The morphological abnormalities were significantly (P<0.01) lower in BHT and taurine than ascorbic acid and control during preservation up to 72h at 4°C. The percent HOST reacted spermatozoa was significantly (P<0.01) higher for BHT and significantly lower (P<0.01) for ascorbic acid than control. The percent intact acrosome was significantly (P<0.01) higher in all the extenders containing antioxidants than control. In conclusion, Tris-citric acid-fructose extender (TCFEY) was found best in maintaining the quality of ejaculated ram spermatozoa during preservation for 72h at 4°C. BHT and Taurine were found to improve the quality of ejaculated ram spermatozoa during preservation for 72h at 4°C.ThesisItem Open Access Cryopreservation of ram epididymal spermatozoa using different extenders and levels of glycerol(SKUAST Kashmir, 2014) Touqeer Ahmed; Rafiqul IslamTesticles of adult healthy slaughtered rams were collected from slaughter houses in and around Srinagar city. Upon reaching the laboratory, the testicles were weighed, cauda epididymis were separated from them and used for recovery of spermatozoa by incision method. Then progressive motility of the spermatozoa in the recovered samples from each cauda epididymidis was determined and samples showing sperm motility ≥70 % were pooled and used as pool sperm samples in the different phases of this study. The quality of spermatozoa on the basis of percentage of sperm motility, live sperm, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa was evaluated immediately after extension in the particular extenders (pre freeze) and at post thaw under each phase. The present study was conducted in three different phases viz. Phase I: Effect of different extenders viz. Tris citric acid fructose (TCF), Tris citric acid glucose (TCG), Egg yolk citrate fructose (SCF) and Egg yolk citrate glucose (SCG) on the quality of cauda epididymal spermatozoa during cryopreservation and post thaw, phase II: Effect of washing on the post thaw quality of cauda epididymal spermatozoa (P1: unwashed, P2: washed) and phase III: Effect of level of glycerol i.e. 4% (G4), 6% (G6) and 8% (G8) on the post thaw quality of ram cauda epididymal spermatozoa. In phase I, the percent sperm motility was significantly (p0.05) for TCF extender both at pre freeze and post thaw than other extenders. The percent intact acrosome was significantly higher (P0.05) in P1 than P2. However, the post thaw percent HOST reacted spermatozoa was slightly higher (P>0.05) for P2 than P1. In phase III, the percent sperm motility at post thaw was significantly higher (P0.05) in G8 than other levels of glycerol (G4 and G6). The percent HOST reacted spermatozoa was significantly higher (P<0.05) for G8 and G6 than G4 at pre freeze. At post thaw, the percent HOST reacted spermatozoa was significantly higher (P<0.01) for G8 than both G6 and G4. In conclusion, Tris-citric acid-fructose extender (TCF) was found best in maintaining the quality of ram cauda epididymal spermatozoa during cryopreservation. Washing of cauda epididymal spermatozoa has no significant adverse effect on the quality during cryopreservation. Therefore, this processing method can be applied wherever necessary before the extension of the recovered spermatozoa sample in different extenders. Glycerol at the rate of 8% should be used as cryoprotectant in TCF extender for freezing of ram cauda epididymal spermatozoa.
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