The effect of thermal stress on superovulatory response and embryo production in Bharat Merino ewes

Small Ruminant Research 55 (2004) 57–63

The effect of thermal stress on superovulatory response and embryo production in Bharat Merino ewes S.M.K. Naqvi∗ , V.P. Maurya, R. Gulyani, A. Joshi, J.P. Mittal Division of Animal Physiology, Central Sheep and Wool Research Institute, Avikanagar, via Jaipur, Rajasthan-304 501, India Received 15 May 2003; received in revised form 17 February 2004; accepted 17 February 2004

Abstract This experiment was conducted to observe the effect of thermal stress during estrous cycle on superovulatory response and subsequent embryo production in Bharat Merino ewes (fine wool crossbred developed for the tropical environment). Fourteen cycling, multiparous ewes were randomly allocated to two groups (n = 7/group). The animals in Group-I were maintained in a shed from 10.00 to 16.00 h while animals in Group-II were exposed to the elevated temperature for a period of 6 h (10.00–16.00 h) for 4 weeks. Animals grazed on natural pasture of Cenchrus ciliaris during the morning (7.00–10.00 h) and evening hours (16.30–19.30 h), and drinking water was offered once daily at 16.30 h. Daily meteorological observations were recorded during the trial. Superovulation was induced in all the ewes using FSH (ovagen 5.4 mg in eight injections) and PMSG (200 IU). Estrus in the ewes was synchronized with 2 doses of PGF2␣ (10 mg) 10 days apart. Ewes demonstrating estrus were hand mated with the aid of Bharat Merino rams. Ovarian examination and embryo/ova recovery were performed using laparoscopy on days 3–5 following mating. All the ewes exhibited estrus within 48 h after the second PGF2␣ injection. Ewes in Group-I exhibited estrus earlier (25.5 ± 1.1 h) and the estrus period was longer (37.7 ± 1.6 h) than ewes exposed to thermal stress, i.e. 30.6 ± 1.2 and 31.7 ± 3.6 h, respectively. No significant difference were observed for the two groups with respect to the superovulatory response and embryo/ova recovery rate. The ewes exposed to thermal stress yielded relatively poor quality embryos. The results indicate that thermal stress could adversely affected the quality of the embryos in Bharat Merino sheep during the preovulatory period. © 2004 Elsevier B.V. All rights reserved. Keywords: Sheep; Synchronization; Estrus; Embryo; Superovulation

1. Introduction The majority (69%) of sheep in India exists in the arid and semi-arid regions and are generally subjected to high ambient temperatures and a limitation to feed. In this environment, sheep have to adapt to maintain homeothermy. Animals exposed to hot environments manifest a significant increase in ∗ Corresponding author. Tel.: +91-1437-220165; fax: +91-1437-220163. E-mail address: [email protected] (S.M.K. Naqvi).

physiological responses to counter-balance the heat stress (Hooda and Naqvi, 1990). Animals, that can maintain their physiological responses within normal limits under stressful environmental conditions may be considered, adapted to that environment and hence, may be worth rearing commercially (Mittal and Ghosh, 1979). Nevertheless, in tropical zones animals have been produced/developed through the infusion of exotic germplasm derived from temperate regions into native stock. High ambient temperatures influence these animals the most and limit their productive potential. The reproductive efficiency of

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sheep is known to be adversely affected by hyperthermia (Thwaites, 1971; Sahani et al., 1976; Sawyer, 1979). Thermal stress is also known to influence the superovulation response in sheep (Gordon, 1997) and cattle (Hansen et al., 2001; Alfujairi et al., 1993; Gordon et al., 1987; Monty and Racowsky, 1987) in a multiple ovulation and embryo transfer programme (MOET). However, information on the effect of thermal stress on the superovulatory response and embryo yield is not available for sheep in the tropics. Therefore, this study was undertaken to examine the superovulatory response and embryo recovery rate in Bharat Merino ewes exposed to elevated ambient temperatures.

2. Materials and methods 2.1. Study site The study was conducted at the Central Sheep & Wool Research Institute, Avikanagar, located in the semi-arid region of sub tropical India at a 75–28◦ E longitude, 26–26◦ N latitude of and an altitude of 320 m above sea level. The annual rainfall in this area ranges from 400 to 600 mm. The minimum and maximum ambient temperatures range from 07 to 37 ◦ C and from 25 to 46 ◦ C, respectively, while the mean relative humidity (RH) varies between 15 and 90%. 2.2. Animals and their management Fourteen 4–7-year-old cycling Bharat Merino ewes weighing 29.8 ± 0.70 kg were randomly divided into two groups (n = 7/group). Bharat Merino is a strain developed for fine wool by crossing native Indian ewes with Rambouillet/Soviet Merino rams and stabilizing the population at 75% exotic inheritance. All ewes were allowed to graze daily in the morning and evening on a Cenchrus ciliaris pasture interspersed with seasonal shrubs, grasses and forbs (Achyranthes aspera, Commelina forskalaei, Eleusine aegypticae and Sorghum helepense). Average biomass yield of pastures plot ranged from 1200 to 1500 kg/hectare. In addition to the pastures, all the ewes received a 200 g concentrate (barley 65%, groundnut cake 32%, mineral mixture 2% and common salt 1%) per head, daily. Drinking water was offered once daily at 16:30 h. The

animals in Group-I (n = 7) were maintained in shed from 10:00 to 16:00 h, while animals in Group-II were subjected to thermal stress (40 ◦ C) in hot chamber for 6 h (10:00–16:00 h) per day for a period of 4 weeks. At night all the ewes were housed in a shed made up of asbestos roofing at height of 2.4 m and opened from sides. Under the shed, the mean ambient temperature and RH were 19.1 ± 0.2 and 72.8 ± 1.3 at 10:00 h and 34.6 ± 0.3 ◦ C and 60.7 ± 0.9% at 16:00 h. The hot chamber was made up of concrete sidewalls with asbestos roof and was large enough to accommodate seven ewes. Hard wooden boards insulated all the inner sides and roof. Provisions was made for the circulation of fresh air and heating with the aid of auto controlled electrical heat blowers. Natural light was provided through double glass air tight windows. The ambient temperature and RH inside the chamber was 40 ◦ C and 58.4%, respectively. No thermal treatment was administered after estrus synchronization and mating. 2.3. Observation of the physiological responses The different physiological responses, i.e. respiration rate (RR, breath/min), pulse rate (PR, beat/min) rectal temperature (RT, ◦ C) were recorded at 8:00 and 14:00 h weekly during the study period. The RR was recorded by the counting flank movements per minute from a distance of 4–5 m without disturbing the ewes. For PR and RT recordings, ewes were gently restrained. PR was measured by palpating the femoral artery and RT was recorded with a clinical thermometer inserted rectally. 2.4. Estrus synchronization and superovulation of ewes Synchronization was induced by administering two intramuscular injections of 10 mg PGF2␣ (PGF) (Lutalyse, Pharmacia, Novaratis India Ltd., India), 10 days apart. Superovulatory treatment commenced 2 days prior to the second PGF injection. Each ewe in the two groups received a total dose of 5.4 mg of ovine FSH (Ovagen, NIADDK-O FSH-17; ICP, Auckland, New Zealand) intramuscularly twice a day (7:30 and 18:30 h) at a constant dose over a 4 day period. Ewes also received a 200 IU PMSG (Folligon, Intervet-Netherlands) intramuscularly at the

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commencement of the superovulation treatment (Ryan et al., 1991; Naqvi and Gulyani, 1998). 2.5. Estrus detection and mating Ewes teased for estrus with the aid of aproned rams (ram : ewe = 1 : 10) of high sexual vigor at 6 h intervals for 3 days commencing at the day of the second PGF2 ␣ injection. Ewes detected in estrus were subjected to mating twice a day (morning and evening) with Bharat Merino rams of proven fertility. Individual rams of proven fertility were assigned to each ewe.

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performed on a warm stage (37 ◦ C) under a stereozoom microscope (Nikon, Japan) at 50× magnification. The fertilization of ova was verified by cleavage. The good quality (transferable) embryos were assessed according to the symmetry of the cells, no shrinkage, vacuolization or lysis. The poor quality embryos had partially degenerated and vacuolated cells. The number of recent ovulation (corpora lutea) and persistent large follicles (LF) were recorded and the ovarian response was estimated by adding the number of CL to the LF. 2.7. Statistical analysis

2.6. Embryo collection Ewes were subjected to laparoscopy for ovarian activity evaluation on days 3–6 after mating. Embryo recovery was performed according to a procedure previously described (Naqvi and Gulyani, 1995; Naqvi et al., 2000; Naqvi et al., 2001). In brief, ewes were fasted for at least 24 h prior to laparoscopy. The abdominal area anterior to the udder was shaved and sprayed with 70% alcohol. Ewes were sedated with xylazine hydrochloride (Xylazine, Indian Immunologicals, India) and locally anaesthetized by infiltration of lignocaine hydrochloride (Xylocaine, SG Pharm, India). Ewes with ≥2 corpora lutea (CL) were deemed as having a superovulatory response and embryos were recovered from these animals. After visualization of the reproductive tract through the laparoscope, the uterine horn was grasped with a Allis Forceps and gently lifted up to the skin surface (McMillan and Hall, 1994; Naqvi and Gulyani, 1995). The uterine horn was held with the fingers and carefully exposed to at least the bifurcation of the two horns. With the aid of the direct flow method, a 20 gauge blunt needle attached to a syringe was inserted into the uterine body near the bifurcation. A hole was also made at the tip of the uterine horn with a 2 mm diameter bone pin (IMV, France) and a polythene catheter (o.d. 3 mm) introduced into the lumen of the horn. Twenty milliliter media (DPBS with 2% BSA) was flushed through the horn using the glass syringe attached to blunt needle. The flushed media was collected in a graduated glass tube to observe the volume. This process was repeated for the other uterine horn. The recovered fluid was transferred into a sterile petri dish and covered. Embryo/ova recovery was

The data on physiological responses was subjected to two way analysis of variance (Snedecor and Cochran, 1980) and Duncan’s multiple range tests was used to test the significance between different mean values (Norusis, 1998). Mean values of estrus and superovulatory responses were analyzed using student’s t-test. Proportions of fertilized and unfertilized eggs, transferable and poor embryos were analyzed using Chi-square test.

3. Results The data on RR, PR and RT of ewes maintained in the shed and hot chamber are set out in Table 1. A significant (P < 0.05) increase in the RR and PR were recorded in both treatments during the study. The magnitude of the increase in physiological responses was significantly higher (P < 0.05) in the animals exposed to thermal stress. Although no significant difference was observed in RT in the period from morning to afternoon in all the animals maintained under normal condition in shed, a significant increase (P < 0.05) in RT was recorded from morning to afternoon in ewes subjected to thermal stress. Both groups of ewes kept in shed and hot chamber environments exhibited estrus during the 72 h after the second injection of PGF. However, the duration of estrus in the control group was significantly (P < 0.05) longer, compared to the ewes subjected to thermal stress. The interval to estrus after the second PGF injection was also significantly (P < 0.05) delayed (Table 2) in ewes exposed to thermal stress compared to control ewes.

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Table 1 Effect of thermal stress on physiological responses (mean ± S.E.) of Bharat Merino ewes Physiological responses

Control (Group-I)

Respiration rate (breaths/min) Pulse rate (beats/min) Rectal temperature (◦ C) a,b,c Means

Heat exposed (Group-II)

Morning

Afternoon

Morning

Afternoon

± 3.1 ± 0.5 38.5b ± 0.1

± 3.0 ± 2.8 38.8a,b ± 0.0

± 3.4 ± 2.6 38.5b ± 0.0

126.5a ± 2.8 96.8a ± 1.3 39.1a ± 0.0

44.5c

73.7b

95.1b

93.2a

42.9c

72.3b

with different superscript in same row differ significantly (P < 0.05).

Table 2 Effect of thermal stress on estrus response (mean ± S.E.) in superovulated Bharat Merino ewes Attributes

Control (Group-I)

Heat exposed (Group-II)

Duration of estrus (h) Interval to estrus (h)

37.7 ± 1.6a 25.4 ± 1.1a

31.7 ± 3.6b 30.6 ± 1.2a

a,b Means

with different superscript in same row differ significantly (P < 0.05).

The effect of thermal stress on ovarian response, ovulation rate and superovulatory response (proportion of ewes with >2 CL) was minimal. In Table 2, the mean superovulatory response in the two treatment groups is set out. The mean number of ovulations and ovarian response were relatively higher in the ewes of Group-I compared to that in the ewes from Group-II. The data pertaining to the recovery of fertilized and unfertilized egg and transferable embryos are also set out in Table 2. The proportion of transferable embryos in relation to the total fertilized ovum recovered were also higher in the Group-I ewes (Table 3). However, The overall fertilization rate was comparable in the

both groups. All the ewes responded to the super ovulation treatment, except one ewe in Group-II. 4. Discussion Native sheep breeds in the hot semi-arid areas when exposed to elevated ambient temperatures or solar radiation during hot summer months are less affected in their physiological functions and production potential compared to non-adapted exotic sheep and crossbreds (Naqvi, 1987; Naqvi and Hooda, 1991; Maurya et al., 1998). The data showed that ewes under hot conditions were under heat stress, as indicated by a greater increase in respiration rate and body temperature. Dutt (1964) reported exposure of Rambouillet cross ewes to severe heat stress from day 12 of the estrous cycle, extended the length of the cycle significantly. In this context, Doney et al. (1973) also reported that severe heat stress could delay the duration of estrus. In the present trial, thermal stress had significant effect on the interval to the onset of estrus. Sawyer (1979) reported high ambient temperatures 1.5–6 days before an

Table 3 Effect of thermal stress on superovulatory response and embryo production and quality in Bharat Merino ewes Attributes

Control (Group-I)

Heat exposed (Group-II)

No. of ewes Ovarian response (CL + LF) mean ± S.E. Ovulation rate (no. of CL/ewe) mean ± S.E. Superovulatory response (no. of ewes >2 CL) Total no. of CL in flushed ewes No. of eggs recovered Fertilized Unfertilized Fertilization rate (%) Transferable embryo no. (%) Poor quality embryos

7 8.86 ± 1.27(7) 8.14 ± 1.17(7) 7 57 35 25 10 71.4 21a (84.0) 4b (1.0)

7 8.0 ± 1.12(7) 6.71 ± 1.10(7) 6 45 38 31 7 71.4 14b (45.2) 17a (54.8)

a,b Values

with different superscript in same row differ significantly (P < 0.05).

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expected estrus significantly reduced the number of ewes displaying estrus .The reason for the delay to onset of estrus could possibly be due to an alteration in the pulsatile release of LH and decrease in estrogen secretion. The normal GnRH release patterns (and subsequent frequency and amplitude of LH pulses secreted from the pituitary) are reduced by exposure to thermal stress (Dobson and Smith, 2000). This results in abnormal ovarian functions and hence cause a delay in the LH surge. Another study indicates the dominant follicle to be susceptible to heat stress (Wolfenson et al., 1997). Furthermore, it has been reported that heat stress alters follicular development and dominance which leads to a decrease in estrogen secretion. Badinga et al. (1993) found follicular dominance to be altered in cows that were heat stressed during the first 8 days of the estrous cycle. However, Gangawar et al. (1965) also reported the duration of estrus to average 20 h in cows maintained under cool conditions, compared to 11 and 14 h for cows maintained in a hot psychrometric chamber and natural summer, respectively. The intensity of estrus was also greater under cool than hot environmental conditions (as in the present study, shed and hot chamber conditions). The estrus response, fertilization rate and neonatal survival may also decrease with heat stress (Mohamed, 1974). In this study, the ewes were subjected to thermal stress prior and during superovulation treatment which in turn had influenced ovulation rate to a lesser extent. The effect of heat stress on superovulation has been recorded to be variable in cattle. Monty and Racowsky (1987) reported no influence on superovulation response in Holstein cows, while adverse effect were observed by other workers (Gordon et al., 1987; Alfujairi et al., 1993). The reasons for the reduced ovulation rate are not clear from this study. The decrease in ovulation rate was less in the current study, and can be attributed to the adaptability of Bharat Merino ewes to hot-arid environments. These animals have been previously selected on the merits of their survival rate in hot semi arid environments. Further, no seasonal variation (between autumn and spring) was recorded in superovulatory response of Bharat Merino ewes (Naqvi et al., 1998) and Chios sheep (Samartazi et al., 1995) in semi-arid areas. The superovulatory response of ewes in this study was not significantly affected by thermal stress, presumably as the individual potential for ovulation was

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maximized by the exogenous gonadotrophin treatment. Putney et al. (1988) and Alfujairi et al. (1993) reported a negative effect of hot summer on ovulation rate, total ova/embryos and quality of embryos in cows. Similarly, Gordon et al. (1987) registered a highly significant difference in values recorded for Holstein cows treated for superovulation during mid summer and winter/spring. In the present study, the fertilization rate of the ewes was not affected by thermal stress. Putney et al. (1989) in exposing Holstein cows to elevated ambient temperature (42 ◦ C for 10 h) during the periovulatory period increased the incidence of retarded and/or abnormal embryos. Similarly, Dutt (1963) reported elevated ambient temperature 24 h prior to fertilization to have no effect on the fertilization rate but increase the incidences of embryonic abnormalities. Similar results have been found in mice by Baumangartner and Chrisman (1987), who indicated that heat stress prior to ovulation did not interfere with oocyte fertilization but resulted in extensive developmental embryonic retardation. The effect of thermal stress on ovulation rate and ovarian response was minimal in this study. This suggested two possible ways by which thermal stress could compromise follicular development: (1) either the magnitude of thermal stress was not high enough as to disrupt folliculogenesis or (2) adaptability of Bharat Merino sheep to high ambient temperature is sufficient. Bharat Merino sheep evolved by crossing and selection over years in semi-arid hot areas, and thus is acclimatized to hot environments. Moreover, ewes have enough opportunity to compensate for the influence of thermal stress, if any, during the more comfortable hours of night and early morning. The influence of thermal stress on the quality of embryos produced from the sheep was evident. The incidence of embryonic abnormalities as presented by (proportion of poor quality embryos) produced from the ewes exposed to thermal stress was higher when compared to the ewes kept under the control environment. The ewes in this trial were not exposed to thermal stress during embryonic development, thus poor quality embryos was due to heat stress during the follicular phase. This could indicate that thermal stress during folliculogenesis lead to ovulation of low quality oocytes with decreased developmental competence. Thermal stress is reported to decrease follicular growth

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(Badinga et al., 1993) and 17-␤ estradiol concentration in the follicular fluid (Wolfenson et al., 1997) in dairy cattle. These consequences may lead to a disruption in the follicular development and influence the oocyte ability for either fertilization or embryonic development. The effect of heat stress on oocyte competence can affect the quality of the embryos. In this study, the heat stress (40 ◦ C for 6 h) treatment was given only during oocyte development (follicular phase of estrous cycle and pre ovulatory period) with minimal influence was recorded on ovulation rate with no effect on fertilization.

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