Anil Joshi

Development and application of ovine reproductive technologies: an Indian experience

Sheep play an important role in the Indian economy by providing employment to a large population of marginal and landless farmers. The production from native breeds is relatively low due to their poor reproductive efficiency. Embryo transfer technology can be utilised for faster multiplication of elite animal to increase the genetic gain. A great deal of research is involved to overcome the constraints in the technology, i.e. expensive and complicated nature of the technology and low success rate. In order to avoid surgical involvement, procedures of laparoscope aided embryo collection and transfer have been developed. Although the use of FSH of ovine origin has given more consistent superovulatory response, but it is still too low to get sufficient numbers of progeny from a donor ewe. The progress made in cryopreservation of ram semen has opened the possibility for conservation and utilisation of frozen semen of elite rams in sheep improvement programme. The lambing rate obtained after laparoscope aided intrauterine artificial insemination with frozen semen is encouraging but the impetus is now to develop the non-invasive transcervical insemination technique.

Effect of induced body condition score differences on physiological response, productive and reproductive performance of Malpura ewes kept in a hot, semi‐arid environment

This study was undertaken to study the influence of induced body condition score (BCS) differences on physiological response, productive and reproductive performance of Malpura ewes to optimise BCS for these ewes for maximising production making it economically viable. The study was conducted for a period of 1 year using thirty healthy Malpura ewes (2-4 year old). The animals were randomly divided and different BCS was induced within three groups named Group I (BCS 2.5; n = 10), Group II (BCS 3.0-3.5; n = 10) and Group III (BCS 4.0; n = 10). The parameters included in the study were allometric measurements, physiological response, wool yield and reproductive performance. BCS had a significant influence on allometric measurements, respiration rate and different reproductive parameters studied, while wool production differed significantly during spring and non-significantly during autumn. The results revealed that the reproductive performance of Malpura ewes with a BCS of 3.0-3.5 was better in comparison with the groups with lower and higher BCS. It may be concluded from this study that an active management of breeding sheep flock to achieve a BCS of 3.0-3.5 may prove to result in an economically viable return from these flocks.

Effect of freezing temperature, at which straws were plunged into liquid nitrogen, on the post-thaw motility and acrosomal status of ram spermatozoa

The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.

Cervical penetration and transcervical AI of tropical sheep (Malpura) at natural oestrus using frozen-thawed semen

Abstract This study comprising of two trials was undertaken in ewes exhibiting natural oestrus to evaluate a simple transcervical artificial insemination technique suitably modified for use in tropical sheep. Twenty four adult multiparous Malpura ewes which had lambed 6 to 12 months prior to the experiment were utilized for two oestrous cycles during the autumn breeding season. The cervical penetration ( n =47) was conducted in both first ( n =24) and second ( n =23) trial ≤6 to 18 h following onset of oestrus, while lambing rate was assessed in the second trial ( n =23) following deposition of frozen-thawed semen having >70% mean post-thaw motility at os, mid cervix or uterus. The overall success achieved in cervical penetration was 44.7%. There was no difference in cervical penetration rate at ≤6 and 18 h after detection of oestrus (46.1% vs. 42.8%, respectively). A mean lambing rate of 22.7% was recorded in ewes inseminated using this technique. Mid cervical and transcervical insemination with frozen semen resulted in similar lambing rate (28.5 vs. 22.7%, respectively). However, no lambing was achieved on os-cervical insemination. Further efforts are needed to determine factors controlling the cervical penetration and conception rate following artificial insemination with frozen semen.

Sperm Motion Characteristics of Garole Rams Raised for a Prolonged Period in a Semi-arid Tropical Environment

The Garole is a prolific but less well known and rare breed of small sheep found in the hot and humid Sunderban region of West Bengal. An ability to breed throughout the year and to graze in knee-deep water, resistance to foot rot and a strong mothering instinct are some of the special features of this breed. Garole rams could provide germplasm to incorporate prolificacy traits by artificial insemination of the nonprolific sheep breeds found in abundance in the semi-arid and arid tropical climates of India. The aim of the present study was to evaluate the semen production by Garole rams maintained in a semi-arid climate for three years and to objectively assess their semen quality by a computer-assisted sperm analysis technique. The donor rams were randomly selected each year from the original flock procured from their natural habitat or from the offspring born at the Institute farm. Semen was collected weekly for three weeks each autumn for three consecutive years from 8 rams each year. The overall means (SD) of the traits that did not differ significantly with age or year were volume, concentration, curvilinear velocity, average path velocity, amplitude of lateral head displacement, beat frequency, motility and the percentages of rapid motile sperms and of slow motile sperms. The age of the rams had a significant effect (p<0.05) on the straight-line velocity but this was not significantly affected by the length of exposure to the semi-arid climate. However, the age and year had significant effects (p<0.05) on linearity, straightness and the percentage of medium motile sperms. It was concluded that Garole rams are capable of producing good-quality semen even after a prolonged period of exposure to a semi-arid tropical climate.

Effect of Controlled and Uncontrolled Rate of Cooling, Prior to Controlled Rate of Freezing, on Motion Characteristics and Acrosomal Integrity of Cryopreserved Ram Spermatozoa

A programmable cell freezer provides ideal cryobiological conditions for controlled-rate cooling and freezing of ram spermatozoa. The purpose of this study was to investigate the effects of controlled (Group 1) and uncontrolled (Group 2) cooling conditions prior to programmable freezing of ram semen on post-thaw sperm motion characteristics and acrosomal integrity of ram spermatozoa. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 × 10(9) spermatozoa per milliliter with Egg yolk-TEST-glycerol extender, and packaged in 0.25 mL straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25°C to 5°C at the rate of -0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5°C to -125°C at the rate of -25°C per minute. Thawing of straws was done at 50°C for 10 s and the quality of frozen-thawed spermatozoa was objectively assessed by using sperm motility analyzer. Thawed samples were also evaluated for acrosomal integrity after staining the dried semen smears with Giemsa stain. The average post-thaw motility of straws was significantly higher (P < 0.05) in samples frozen after controlled cooling, compared with samples frozen after uncontrolled rate of cooling. The percent of spermatozoa with normal acrosome was also significantly (P < 0.05) higher in Group 1, compared to Group 2. The results indicate that controlled-rate cooling has a significant effect on post-thaw motility and acrosomal integrity of frozen-thawed ram spermatozoa, compared to uncontrolled-rate cooling prior to programmable freezing.

Production of prolific microsheep by embryo transfer into large non-prolific sheep

The Garole is a prolific breed of microsheep that possesses the FecB gene, which increases ovulation rate. The purpose of this study was to compare embryo production by multiple ovulation in seven Garole ewes with that in seven normal size, non-prolific Malpura ewes, and assess the influence of the large body size of Awassi crossbred recipient ewes on the birthweight of Garole lambs. Oestrus was synchronised with two intramuscular injections of 7·5 mg prostaglandin F2α administered 10 days apart. The donor ewes were superovulated by the use of pregnant mare serum gonadotrophin and follicle-stimulating hormone. The onset and duration of oestrus were similar in both breeds. The Garole donors had higher total mean (se) ovarian responses (15·6 [3·6] v 9·1 [2·3]), ovulation rate (13·6 [3·1] v 8·4 [2·2]) and produced more transferable embryos (6·0 [3·5] v 4·0 [0·9]) than the Malpura donors, but the differences were not statistically significant. The Garole lambs produced by embryo transfer were on average 57·8 per cent heavier at birth than contemporary Garole lambs produced by natural mating.

Production of 24 transferable embryos in one flush by a Garole microsheep

FIG 2: Ovaries of a superovulated Garole ewe, showing multiple ovulations An adult parous Garole ewe weighing 12·5 kg, belonging to a flock that was procured from the Sunderban area in October 2000 and raised under a semi-intensive management system at the institute’s farm, was induced for superovulation in late autumn 2004 under the multiple ovulation embryo transfer programme being conducted at the institute. Oestrus was synchronised by the administration of two intramuscular injections of 10 mg prostaglandin F2α (PGF2α) (Lutalyse; Pharmacia), 10 days apart. Superovulatory treatment commenced three days before the second PGF2α injection: the ewe received a total dose of 5·4 mg of ovine follicle-stimulating hormone (Ovagen; ICP), intramuscularly twice a day, at 07.30 and 18.30, at a constant dose over a four-day period. A dose of 200 IU pregnant mare serum gonadotrophin (Folligon; Intervet) was also administered intramuscularly at the start of the superovulation treatment. Oestrus in the ewe was detected with the aid of an aproned ram of high sexual vigour at six-hourly intervals for three days, beginning on the day of the second PGF2α injection. The ewe was then subjected to mating twice a day, in the morning and evening, with a Garole ram of proven fertility. The ovarian response in terms of the number of ovulations was determined by laparoscopic examination four days after mating. Surgical embryo collection was performed as described by Naqvi and others (2001). The ewe was fasted for at least 24 hours before the surgical intervention. The abdominal area anterior to the udder was shaved and sprayed with 70 per cent alcohol, and the ewe was sedated with xylazine hydrochloride (Xylazine; Indian Immunologicals) and locally anaesthetised by the infiltration of lignocaine hydrochloride (Xylocaine; AstraZeneca). After visualisation of the reproductive tract through the laparotomy, the uterine horn was carefully exposed to at least the bifurcation of the two horns. The flushing medium, 20 ml Dulbecco’s phosphate buffered saline, pH 7·5 (± 0·3) at room temperature, supplemented with 2 per cent bovine serum albumin (Sigma Chemical) was introduced into the base of the uterine horn using a blunt Jelco needle attached to a syringe. The medium was flushed towards the tip, where it was collected through a polythene catheter (outer Production of 24 transferable embryos in one flush by a Garole microsheep