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INCLUSION OF CIDR IN SUPEROVULATORY PROTOCOL ON EMBRYO RECOVERY IN KANGAYAM COWS
(2021) PERIYANNAN M; TANUVAS; SELVARAJU M; RAVIKUMAR K; RAJA A
In cows, superovulation should allow the production of a maximum number of transferable embryos capable of establishing pregnancy after transfer, maintaining normal embryonic development and resuming pregnancy with the expulsion of healthy progenies of genetic superiority. A total of 16 Kangayam cows were selected and randomly divided into two equal experimental groups viz., group I and II. All the donor cows were treated with two doses of 500 μg PGF2α (i.m.) at an interval of 11 days. In group I donor cows, CIDR was inserted intravaginally on day 7 after estrus. Two mg of estradiol benzoate was injected intramuscularly at the time of CIDR insertion to all the donor cows. In group II donor cows, injection estradiol benzoate 2 mg was injected intramuscularly on day 7 after the estrus without CIDR insertion. In both the groups, from day 11, 50 mg of NIH FSH was given (i.m.) during morning and evening (twice daily) to each donor cow for a period of 4 days at an interval of 12 hours. On day 13, 500 μg of PGF2α was administered (i.m.) during morning and evening followed by CIDR was removed 12 hours after the second PGF2α injection. At 12 hours after the last FSH injection, 20 μg of inj. GnRH was given to each donor cow (i.m.). A total of 3 AIs were done at an interval of 12 hours starting at 12 hours after the GnRH injection. The embryos were collected from superovulated donors on day 7 after 1st AI by non-surgical transcervical method. A Foley catheter was fixed at the anterior 1/3rd of the uterine horn and the balloon of Foley catheter was inflated with 8 – 12 ml of air. The right uterine horn was thoroughly flushed for 3-5 times using flushing media and the same procedure was repeated for the opposite horn.
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SPERM VIABILITY AND FERTILITY RATE OF KANGAYAM BULL SEMEN EXTENDED WITH DIFFERENT DILUENTS
(2021) ELAMURUGAN K; TANUVAS; EZAKIAL NAPOLEAN R; SELVARAJU M; MURALI N
Awareness of artificial insemination (AI) in Kangayam and other native cows among farmers of Tamil Nadu state is increasing due to non-availability of pedigreed and suitable breeding bulls and the Jallikattu fest. Identification of suitable extender for cryopreservation of native bull semen is the need of hour in native animal breeding. Kangayam bull No NK 14 aged 5 years was utilized for the study. The semen was collected twice in a week and each time two ejaculates were collected. Then they were immediately transferred to semen processing laboratory and kept at 34° C in the water bath. The semen collected from the bull into the sterile disposable plastic graduated tubes was immediately subjected to macroscopic, microscopic and biochemical analysis. A two μl of neat semen sample was diluted with 400 μl of Tris solution. After dilution, two μl of sample was loaded in one chamber of prewarmed Leja slide and kept in microscopic stage of CASA and all the sperm kinetic characters were analyzed. Tris- egg - yolk glycerol (TEYG) (group I), soya lecithin based extender (group II) and liposome based extender (group III) were prepared on the day of semen collection and kept in water bath at 34° C for processing of semen. Initially the semen sample was diluted at 1:1 ratio with the corresponding extender (TEYG or soya lecithin based or liposome based extenders) according to the group. The 1:1 diluted semen samples from each group were kept at 22° C for 7 minutes in the laminar air flow. During this time the temperature of semen sample was reduced from 34° C to 22° C. After reaching 22° C, the 1:1 diluted semen samples from each group was finally extended with respective semen extender as per the dilution rate.
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ESTRUS SPECIFIC PROTEIN PROFILE IN SALIVA, CERVICO-VAGINAL FLUID (CVF) AND URINE DURING STANDING ESTRUS IN SILENT ESTRUS BUFFALOES
(2022) PUGAZHARASI C; TANUVAS; KRISHNAKUMAR K; MEIGNANALAKSHMI S; JAISHANKAR S
Buffaloes have high production potential but their reproductive efficiency is often compromised by delayed puberty, late sexual maturity, silent estrus and poor estrus expression which limits the reproductive potential. Silent estrus is one of the major problems in buffaloes which causes huge economic loss to the farmers. Hence, development of appropriate diagnostic assay for detection of silent estrus in buffaloes are thrust area of research and need of the hour too. Saliva and other easily accessible body fluids such as cervico-vaginal fluid and urine have been widely employed in the recent years for protein profiling to identify proteins for developing useful non-invasive biomarker for various diagnostic applications. Therefore, the present study is designed to study the ovarian steroids, cortisol profile, estrus specific protein profile in saliva, cervico-vaginal fluid (CVF) and urine during standing estrus in silent estrus buffaloes. Eighteen healthy non-pregnant graded Murrah buffaloes at PGRIAS, Kattupakkam were selected for this study and synchronized using progesterone device. The selected buffaloes were randomly allotted in to three groups viz. Group I (Induced estrus), Group II (Regular estrus) and Group III (Silent estrus). The blood samples were collected during proestrus, pre-estrus, estrus and diestrus from all the groups for estimation of hormone profile by RIA. The saliva, CVF and urine samples were collected for total protein estimation by Bradford assay and protein profiling using SDS-PAGE.
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EVALUATION OF OXIDATIVE STRESS MARKERS AS PROGNOSTIC INDICATORS FOR OPEN CERVIX PYOMETRA IN BITCHES
(2022) NIDHIYA JOY K; TANUVAS; METHAI A; KRISHNAKUMAR K; VAIRAMUTHU S; TANUVAS
Pyometra is considered as a diestrual disorder of aged intact bitches, but the cases of pyometra have been reported in dogs of breeding age. In such cases restoring the breeding value of animals should be given importance rather than opting for usual ovariohysterectomy. The present study was carried out in Small Animal Gynaecology and Obstetrics Out Patient Ward (SAC-OG-OP) of Madras Veterinary College Teaching Hospital and bitches aged between 2 and 6 years of age. Eighteen bitches diagnosed with open cervix pyometra were selected and grouped randomly into three groups. Diagnosis of the condition was done based on history, clinical signs, hematology, serum biochemistry, ultrasonography, radiography and Vaginal Exfoliative Cytology (VEC). In Group I (n= 6), bitches were treated with a combination cloprostenol @ 5μg/ kg bwt SC and Cabergoline @ 5μg/kg bwt PO SID for 7 days; group II (n= 6) bitches were treated with Dinoprost tromethamine intravaginally at the dose rate of 150 μg/ kg bwt for 7 days; group III (n= 6) bitches were treated surgically by ovariohysterectomy. Six apparently healthy bitches in dioestrus were kept as control group (Group IV). Antibiotic was selected based on Antibiotic Sensitivity Test (ABST). Serum samples and whole blood samples were collected from each bitch at weekly intervals and haematological, biochemical, electrolyte as well as oxidative stress markers were estimated. Response to the treatment was assessed based on clinical signs, haematology, serum biochemistry, ultrasonography, radiography and vaginal exfoliative cytology.
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IDENTIFICATION OF OESTRUS SPECIFIC PROTEINS FROM SALIVA AND CERVICO VAGINAL FLUIDS IN OESTROUS AND SILENT OESTROUS BUFFALOES
(2021) Sathishkumar S; TANUVAS; Cecilia Joseph; Sarath T; Senthilkumar TMA; Vijaya Bharathi M
confirmation of oestrus in cattle but none of the methods have succeeded to Buffaloes have high productive potential but poor manifestations of oestrus signs and silent oestrus act as a major constraint in buffaloes affecting their reproduction. Hence, accurate and efficient detection of oestrus is a key factor for right time of artificial insemination to achieve successful conception rate in buffaloes. There are several detection tools used for detection and overcome the problem of silent oestrus in buffaloes. Recently proteomics approach has gained much attention to identify the presence of large number of bio molecules in different body fluids. In recent years saliva and cervico vaginal fluid are extensively used for profiling of proteins to identify proteins associated with various physiological and disease conditions, mostly in humans and up to certain extent in cattle and buffaloes. In this study eighteen pleuriparous buffaloes aged 4 to 7 years with good body condition were selected and divided into group I (regular oestrus), group II (silent oestrus) and group III (anestrus) buffaloes. Each group carries six animals and saliva was collected at proestrus (day -3), oestrus (day 0), and diestrus stage (day 7) in group I and group II buffaloes and during anestrus in group III buffaloes. CVF was collected only at oestrum stage in group I and group II buffaloes. Intensity of oestrus, protein concentration and protein profiling of buffaloes were studied by Bradford method and SDS - PAGE analysis. The animals which scored 11-15 points on a scale of 15 were with intense in their exhibition of oestrual signs. The animals which scored 6-10 points were with intermediate signs and the animals with weak oestrus signs scored from 0 to 5. In this study in group I, 16.66 per cent (1/6) animal showed weak, 16.66 per cent (1/6) showed intermediate and 66.68 per cent (4/6) showed intense oestrus signs. In group - II of silent oestrus animals, 16.66 per cent (1/6), 83.34 per cent (5/6) and 0.00 per cent (0/6) of animals showed weak, intermediate and intense oestrus signs, respectively. None of the animals in group III showed oestrus signs. The protein concentration in the saliva of oestrus buffaloes of group I were 0.48 ± 0.01, 0.56 ± 0.03 and 0.48 ± 0.03 during proestrous, oestrous and diestrus of the cycle respectively. In the group II animals of silent oestrus buffaloes, the protein levels were 0.50 ± 0.04, 0.54 ± 0.05 and 0.41 ± 0.01 during proestrous, oestrous and diestrus of the cycle, respectively. The protein concentration of CVF during oestrus was 0.35 ±0.18 and 0.55 ± 0.07 in regular and silent oestrus buffaloes, respectively. The concentration of proteins in saliva of regular cycling and anestrus buffaloes of group I and III was 0.56 ± 0.03 and 0.45 ± 0.08 respectively. The concentration of protein in saliva of silent oestrus buffaloes and anestrus buffaloes was 0.54 ± 0.05 and 0.46 ± 0.08 respectively. The protein concentration was not significantly different between the regular and anestrus group I and III buffaloes and silent and anestrus group 11 and group III buffaloes. However, the concentration of protein in silent oestrus and regular oestrus was slightly higher than the anestrus buffaloes. The SDS-PAGE analysis showed proteins of molecular weight 150, 110, 80, 40 kDas during proestrus; 150, 120, 70 and 50 kDas during oestrus; 250, 150, 90, 50 and 37 kDas during diestrus in group I buffaloes. Similarly, proteins of molecular weight 250, 150, 100, 80 and 25 kDas during proestrus; 150, 100, 75 and 50 kDas during oestrus and 250, 100,75 and 25 kDas during diestrus were observed in SDS - PAGE analysis in group II silent oestrus buffaloes.The SDSPAGE analysis of CVF showed proteins of molecular weight of 100 and 75kDa in group I buffaloes during oestrus and 250 and 150kDa during silent oestrus in group n buffaloes. Anestrous showed the presence of proteins of different molecular weight with 150, 100, 75, 52 and 30kDa in the SDS-PAGE analysis. It could be concluded from the present study that intensity of oestrus is intense in regular, intermeditae in silent oestrus and weak in anestrus buffaloes. The hormonal and secretary change during the different stages of oestrus cycle is reflected by the changes in the protein in saliva and cervico vaginal fluid. Oestrus specific proteins with different molecular weights were identified interms of different bands size. However further study is warranted to study their role during oestrus in regular and silent oestrus buffaloes for the development of specific diagnostic assay.
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LOW-DENSITY LIPOPROTEIN (LDL) SUPPLEMENTATION TO TRIS-BASED DILUENT AND ITS INFLUENCE ON VITAL AND ULTRA-STRUCTURAL CHARACTERISTICS OF FROZEN-THAWED CANINE SPERMATOZOA
(2021) Rajput Shubhamsingh Chandansingh; TANUVAS; Arunmozhi N; Rangasamy S; Kannan TA
The present research was aimed to study the effect of low-density lipoprotein (LDL) based extender on semen parameters like progressive motility. viability, plasma membrane integrity, acrosome membrane integrity, DNA integrity and mitochondrial membrane potential including ultrastructural characteristics Semen was collected from the dogs with a body condition score of 4-5 (on a scale of 0-9) by digital manipulation. The pre-freeze characteristics of the semen like volume, colour, consistency, concentration, mass motility, progressive motility, viability and total sperm abnormalities were studied. Following which the sperm rich fractions were diluted in the ratio of 1:1 or 1:2 with a TRJS-glucose bound extender containing 20 per cent egg yolk in trial 1 and with TRIS-glucose bound extender containing 8 per cent low-density lipoprotein (LDL) in trial 2 depending upon the sperm concentration, LDL was extracted as per the procedure described by Moussa et al., (2002). Then cryopreservation was done as per conventional method. The effect of LDL supplementation on progressive motility, viability, plasma membrane integrity, acrosome membrane integrity, DNA integrity and mitochondrial membrane potential at 24 h post-thaw were evaluated and compared with Tris-egg yolk extender. Morphological and functional characteristics of spermatozoa with EY and LDL extender at 6 h of equilibration were recorded and was found to be significantly higher for the percentage of viability, plasma membrane and DNA integrity and mitochondrial membrane potential. However, no significant difference was observed for plasma membrane integrity in EY and LDL added semen at 6 h of equilibration. After cryopreservation the semen samples were evaluated for progressive motility, viability, plasma membrane integrity, acrosomal integrity, DNA integrity and mitochondrial membrane potential at 24 hours post thaw and significantly higher difference (p<0.01) was observed in the mean percentage of progressive motility, viability, plasma membrane integrity, acrosomal integrity and mitochondrial membrane potential. No significant difference was noticed in DNA integrity between the semen extended with EY and LDL at 24 h post-thaw, which might be due to more stable nuclear packing and highly condensed with a unique DNA organization. The semen samples diluted with EY and LDL extenders were evaluated for all the above said parameters at 6 h of equilibration and 24 hours post thaw and the result revealed that all the parameters were highly significant between 6 h equilibration and 24 h post-thaw in the semen samples extended with both EY and LDL. This could be because cryopreservation induces lethal intra-cellular ice crystal formation in turn causing damage in the plasma and acrosomal membrane. Cryopreservation also modifies the lipid biochemical organization and pattern. It also changes the biochemical composition and structure of the spermatozoal plasma and acrosomal membrane. Ultrastructural characteristics of fresh semen, semen samples at 6 h of equilibration and 24 h post-thaw by SEM showed that, there was more damage noticed in different sperm regions in samples extended with EY at 6 h and 24 h post-thaw when compared to LDL. TEM micrograph of the canine semen added with EY extender showed damaged acrosome and neck region while the semen extended with LDL showed intact acrosomal membrane and intact head region with some damages inflicted in the plasma membrane. TEM picture also revealed damaged acrosome with leakage of enzymes and severe vesiculations and damage of the plasma membrane in semen extended with EY while in semen added with LDL extender, head and mid-piece were intact in TEM micrograph. In conclusion, addition of LDL at 8 per cent concentration in Tris-glucose bound extender could be a better alternative to egg yolk for cryopreservation of canine semen as morphological, functional and ultra-structural damages induced by cryopreservation are minimal.
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EXPRESSION PROFILE OF OASl AND MX2 GENES IN PERIPHERAL BLOOD AND LUTEAL CELLS FOR EARLY PREGNANCY DIAGNOSIS IN CATTLE
(2021) Kanaga Devi M; TANUVAS; Krishnakumar K; Sarath T; Tirumurugaan KG
The present study was planned to analyse the expression levels of OAS 1 and MX2 genes in Peripheral Blood Mononuclear Cells (PBMC) and luteal cell culture upon stimulation of Interferon tau (IFNT). 30 cows were selected and divided into 2 groups as group 1 (n^lO) and II (n=20). Group I cows served as control and group 11 cows were synchronized with CIDR + PGF2a protocol in the presence of matured CL. Breeding was carried during the observed estrus in group 1 and 48 and 72 h after removal of CIDR in group II cows. The blood samples were collected on day 0, 7, 14, 18 and 21 post insemination and PBMC harvested was utilized for isolation and synthesis of RNA and cDNA. Quantitative real time PCR was carried out for OASl and MX2 genes and relative expression was calculated by 2’^^ method. Buffalo ovaries with corpus luteum (CL) were collected immediately following slaughter. Mid luteal phase CL was selected and cell culture was carried out and incubated for 72 h. Cultured luteal cells were treated with three early pregnant serum which was collected on 24^ day post Al from group II cows and stored at -80° C. Varying concentrations of Interferon tau (IFNT) as 1, 10 and 100 ng/ml were also exposed to cultured luteal cells and incubated for 48 h and cells were harvested for RNA isolation followed by cDNA synthesis. Quantitative real time PCR was carried out for OASl and MX2 genes and relative expression was calculated by 2 2 -^^CT method. The results concluded that cows treated with CIDR + PGFaa showed 100 per cent estrus induction response and achieved 60.00 per cent of conception rate. Expression of both OASl and MX2 genes in PBMC was significantly higher (P<0.01) on day 14, 18 and 21 in group I and 11 pregnant cows, when compared to group I and 11 non pregnant cows. No significant difference was observed in the relative expression of OASl between different days in non pregnant cows of group I and II and between different days in pregnant cows of group 1 and IL In luteal cell culture, the OASl and MX2 expression was significantly higher (P<0.05) in early pregnant serum and IFNT treated luteal cells when compared to untreated control luteal cells. The OASl and MX2 expression was significantly higher (P<0.()5) in 10 and 100 ng/ml IFNT treated luteal cells when compared to Ing/ml IFNT treated luteal cells. Hence, it could be inferred that the higher expression of OAS1 and MX2 genes during early pregnancy corresponds to the translation of related proteins, which if identified could be used as a bio marker for early pregnancy diagnosis in cattle.
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SILENT ESTRUS IN BUFFALOES: EXPRESSION PROFILE ANALYSIS OF CANDIDATE GENES AND SALIVARY AND CERVICO-VAGINAL FLUID PROTEOMICS
(2021) Suresh Kumar R; TANUVAS; Krishnakumar K; Balasubramanian S; Vijayarani K; Thilak Pon Jawahar K; Balasubramanyam D
The present study was planned to identify the estrus specific protein expression in saliva and cervico-vaginal fluid and estrus specific candidate gene expression in silent estrus graded Murrah buffaloes. In this study, 14 number of graded Murrah buffaloes were selected and divided into two groups as I (n=7) normal estrus, and II (n=7) silent estrus. Group II silent estrus buffaloes were induced estrus with CIDR + PGFia protocol and considered as group III (n=7) estrus induced silent estrus buffaloes. Estrus induction response was recorded as 100 per cent in group n buffaloes induced with CIDR + PGF2a protocol (group III). The intense, intermediate and weak estrus was 85.71, 14.28 and 0.00 per cent in group I, 0.00, 28.57 and 71.43 per cent in group II and 37.14, 42.68 and 0.00 per cent in group III buffaloes. The higher percentage of intense and moderate estrus intensity was observed in group I and group III than group 11 buffaloes. The overall estrus behavioural signs were 44.44, 11.10 and 42.85 per cent in group 1,11 and III, respectively in buffaloes which was significantly lower in group 11 buffaloes. The duration of estrus was 22.71±0.86, 19.00±0.75 and 24.42±1.13 h in group I, II and III, respectively, which revealed that the group II buffaloes differed significantly from other groups. The estrous cycle length was 23.80±0.17, 22.09±0.28 and 23.85±0.90 days in group I, II and III, respectively in buffaloes which was significantly lower in group II than remaining groups.
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CONTRACEPTIVE INTERVENTION BY VAS OCCLUSION USING COPOLYMER STYRENE MALEIC ANHYDRIDE (SMA) » IN MALE RATS AND IN VITRO STUDY IN DOGS
(2021) UMAMAGESWARI J; TANUVAS; Balasubramanian S; Krishnakumar K; Vijayarani K; Preetha SP
The non-hormonal, copolymeric, contraceptive agent, Styrene maleic anhydride (SMA) is expected to provide a valuable addition to the current options of canine male contraception. The present study was conducted to determine the in vitro spermicidal action of copolymer SMA on canine ejaculatory spermatozoa and to study the in vivo vas occlusion contraceptive effect of copolymer SMA, followed by its reversal using dimethyl sulphoxide (DMSO) and sodium bicarbonate (NaHCOs) in male Wistar rats. The copolymer SMA was developed by Radical addition-fragmentation chain transfer (RAFT) polymerization in the ratio of 2:1 of styrene and maleic anhydride and characterized by Zeta potential analysis. Matrix assisted laser desorption (MALDI-ToF), Nuclear magnetic resonance spectroscopy (NMR) and Fourier- Transfoim Infrared (FTIR) spectroscopy. The sperm rich second fraction of semen collected from twelve adult dogs was subjected to varying dose levels of copolymer SMA (1.0 and 2.0 mg / 100 pl of DMSO ) in vitro and were microscopically analysed for its motility, viability and acrosomal integrity. A significant decline in motility and viability percentage and increase in acrosomal damage of ejaculatory sperm cells were noticed in both 1.0 and 2.0 mg of copolymer SMA treated canine spermatozoa at different time intervals viz. 0, 15, 30, 45 and 60 min when compared to the DMSO treated and untreated control. Both 1.0 and 2.0 mg of copolymer SMA showed 100 per cent spermicidal effect on spermatozoa at 60 min. Hence, all further in vitro studies were carried out using the 1.0 mg dose of SMA. The morphological, topological and ultrastructural changes of canine spermatozoa induced by copolymer SMA (Img/lOOpl of DMSO) were examined using Atomic force microscopy (AFM), Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM). As a result, deleterious changes were observed in the acrosomal and plasma membrane of canine spermatozoa treated with copolymer SMA and no such changes were detected in DMSO and untreated control group of canine spermatozoa. In vivo vas occlusion trials were conducted in experimental groups (G II to G V) of Wistar rats using 5mg of copolymer SMA dissolved in lOpl of DMSO injected intravasally while, only lOpl of DMSO was injected in sham control rats (G I). After 15-19 days post mating, 75 per cent of female rats mated with sham control males and 17, 8, 0 and 0 per cent of the female rats mated with vas occluded group II, HI, rv and V male rats, respectively were found pregnant on ultrasound examination. However, no significant differences were noticed in the sperm count, motility, viability and abnormality percentage of epididymal spermatozoa between sham operated group and SMA treated groups of rat. AFM, SEM and TEM studies also did not show any morphological changes in the head, mid piece and tail regions of epididymal spermatozoa. In vivo vas occlusion reversal experiment was also carried out in twelve Wistar rats (G VI) using solvents viz., DMSO and 5 per cent NaHCOj. At 60 days of post-reversal period, fertility trials showed 67 and 83 per cent restoration of fertility in DMSO and NaHCOs reversed group of rats, respectively. SEM studies showed that the vas lumen was occluded with white, amorphous substances which anchored into the vas mucosal wall by invagination and blocked the patency. Following vas occlusion reversal, vas lumen regained its patency with loss of SMA copolymer plug in the vas lumen and complete regeneration in the vas epithelium was seen. All other parameters viz. haematology, serum biochemistry and testosterone remained unaltered in vas occluded and reversal rats. Thus, use of copolymer SMA as vas occlusive agent was found to be safe, effective and could provide hope as a reversible male contraceptive in canine.

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