Ovarian status in fat-tailed Syrian Awassi ewes during different reproductive stages and hormonal treatments monitored by laparoscopy and progesterone concentrations

Abstract

A study was conducted on seven Syrian Awassi ewes subjected to three treatments: (1) outside the breeding season without any other treatment applied (O + UT group), (2) outside the breeding season but treated with intravaginal sponges (flugestone acetate: [FGA]) only (O + FGA) and (3) inside the breeding season but treated with both FGA and equine chorionic gonadotropin (eCG) (I + FGA + eCG group). Parameters of the ovarian activity in each ovary were monitored by a laparoscope. The progesterone concentrations in blood serum were determined by a radioimmunoassay technique. Diameters of the ovaries and numbers of ovarian follicles were significantly higher (p<0.05) in the ewes treated with FGA. The mean diameters of the ovaries were 1.24, 1.46 and 1.55 cm for ewes in the O + UT, O + FGA and I + FGA + eCG groups, respectively, with no significant differences (p>0.05) in the diameters between the right and left ovaries within the same group. The mean numbers of the <3 mm and 3–5 mm follicles were 5.8 and 0.37, 10.6 and 1.4 and 9.9 and 1.9 follicles for ewes in the O + UT, O + FGA and I + FGA + eCG groups, respectively. Unlike the other two groups, there were no follicles larger than 4 mm found in the ovaries of ewes in the O + UT group. However, follicles larger than 5 mm were found only in some ovaries of the I + FGA + eCG group. No corpora lutea (CL) were found in the ovaries of ewes in the O + UT group. Functional CL were formed in ovaries of ewes in the O + FGA and I + FGA + eCG groups. The latter treatment produced significantly higher numbers, larger sizes and longer lifespans of CL (numbers: 1.6 vs 2.4; diameters: 8.1 vs 8.8 mm and lifespans: 10.7 vs 13.0 d, respectively). Likewise, the mean progesterone concentration during the luteal phase was significantly higher (p<0.05) in the latter than in the former group (20.79 vs 10.23 nmoL l⁻¹, respectively). There were no adhesion formations in the studied organs, or any mortality case among ewes following repeated laparoscopic observations. Therefore, laparoscopy could be safely and repeatedly applied on the same ewe without health problems.

Keywords:

• Ovary
• corpus luteum
• laparoscope
• progesterone
• Syrian Awassi ewe

Introduction

In reproduction, laparoscopy is considered as an accurate method for monitoring the follicular dynamics (Sosa et al. 2010), the status of the corpus luteum (Cognié et al. 2007), determination of ovulation rates (Abella et al. 2005), and in artificial insemination (AI) (Emsen et al. 2008) in ewes. Moreover, laparoscopy has been used in ovariectomy in several animal species such as donkeys (Aziz et al. 2008) and bear (Radisic et al. 2010). In addition, laparoscopy has been also widely used in animal disease diagnosis (Webb and Trott 2008), sterilisation (Dutta et al. 2010), and in biopsy of certain organs (Chiesa et al. 2009).

Lambing rates in Sarda ewes inseminated with transcervical AI were 24% compared to 42% using laparoscopy (Fulaschi et al. 1998). Laparoscopic aspiration of mature oocytes from hormone-treated females offered a valuable source of genetic material for breeding programmes (Bainbridge et al. 1999), and for extending the reproductive life of valuable females that have acquired difficulties becoming pregnant by AI (Baldassarre et al. 2007).

The fat-tailed Awassi sheep breed is a local breed in Syria (about 23 million, AODA 2009) and is considered as the most important and dominant triple-purpose breed in the Middle East area. Due to its importance and strong flocking instinct, this breed has been introduced into many countries (e.g. India: Santra and Karim 2001 and Australia: AlJassim et al. 2002). Syrian Awassi sheep are seasonal breeders and mate between June and September (Zarkawi 1997). They have a relatively poor reproductive performance and a low twinning rate (Thomson and Bahhady 1988), with an average duration of pregnancy of 152.5 d (Zarkawi 2001).

The aims of the present study were to in situ characterise the ovaries, ovarian follicles and corpora lutea (CL) in fat-tailed Syrian Awassi ewes during different reproductive stages inside and outside the breeding season, and to examine the ovarian response to the hormonal treatments using a laparoscope and progesterone concentrations.

Materials and methods

Location, experimental animals and hormonal treatments

This study was carried out at the Animal Production Division, in Deir Al-Hajar area, about 33 km south-east of Damascus (33°21′ N, 36° 28′ E; 617 m above sea level). This area is considered as a dry area with an annual rainfall of about 100 mm occurring mainly in December and January, and it is a part of the Syrian steppe region where the majority of sheep population is raised.

Seven intact cyclic fat-tailed Syrian Awassi ewes weighing (mean±SD) 56.0±8.4 kg, and aged between 2 and 6 years were used three times, twice outside (December and March) and once inside (July) the breeding season. Outside the breeding season, the ewes were either untreated (O + UT group), or treated for oestrous synchronisation with intravaginal sponges containing 40 mg of flugestone acetate (FGA, Chronogest®, Intervet International B.V., The Netherlands) for 14 d (O + FGA group). Ewes inside the breeding season were treated with FGA for 14 d and injected intramuscularly at sponge withdrawal with 500 IU of equine chorionic gonadotropin (eCG, Folligon, Intervet International B.V., The Netherlands) (I + FGA + eCG group).

Animal housing and feeding

When there were no measurements, ewes were kept indoors at night and outside for most of the day. Indoors, the animals were offered diets based on barley and wheat straw supplemented by vitamins (High Vet, Safco Vet Products, Damascus). Outside, they had free access to natural available grazing lands (barley, grass, cotton residues). Water and mineral licks (Phosphadin, Al-Shark Vet Products, Damascus) were available ad libitum.

Laparoscopic measurements and preparation of ewes prior to laparoscopy

The laparoscope used (Karl STORZ, Tutlingen, Germany) consisted of a 7 mm telescope, a light source, a glass fibre cable for the light source, 7 and 5 mm trochars, cannulas and grasping forceps. In addition, an air foot pump connected to a filter and a cradle with variable heights and operating angles were also used.

Ewes were laparoscopically examined three times a week for a period of 18 d for the O + UT group and for 21 d for the O + FGA and I + FGA + eCG groups (to cover an oestrous cycle), as the length in the oestrous cycle of Syrian Awassi ewes was estimated to be around 17 d (Zarkawi 2004). Laparoscopic measurements were performed about 24 h after sponge removal. The specific object used has known dimentions (changeable) in which you can measure the ovaries, ovarian follicles and CL diameters.

Prior to laparoscopy, ewes were fasted for about 17 h. On the day of the measurement, ewes were sedated with 0.01 mL/kg body weight of xylazine 2% (XYLA JECT, ADWIA, Egypt) administered intramuscularly and restrained in a cradle in the classical position for laparoscopy (dorsal recumbency). The abdominal area was prepared by shearing the wool around the udder and from about the first 20 cm of the lower belly area by a razor blade. Dirt was removed from the skin with an antiseptic detergent. Ewes were locally anesthesiased using 0.02 mg/kg body weight lidocaine 2% (Oubari Pharma, Syria), administered subcutaneously about 3 cm anterior to the udder, and about 3–4 cm on each side of the mid-ventral line. A very small incision (about 1 cm) was made in the skin by a scalpel to facilitate puncturing the skin and insertion the 7 mm trochar and the cannula holding the telescope and the gas inlet. By means of the telescope and the light source to locate the right place, the skin was punctured using a 5 mm trochar for inserting the cannula holding the grasping forceps. During the puncturing, care was taken to avoid blood vessels. After measurements, the cannulas were removed and a topical antibacterial spray (Alamycin: Oxytetracycline, Norbrook, UK) was applied on the incisions.

Blood sampling and progesterone analysis

Jugular blood samples (10 mL) were collected from ewes using vacutainers prior to each measurement. Serum was harvested by centrifugation and stored at −20°C until assayed. The validated progesterone RIA kits (Coat–A–Count, Siemens, USA) were used. Progesterone levels equalled to or exceeding 3.18 nmol L⁻¹ were indicative of normal luteal function, and levels <3.18 nmol L⁻¹ were indicative of either anoestrus or follicular phase of the oestrous cycle (Zarkawi 1997).

Parameters and statistical analysis

The following parameters were measured:

•	The diameter of each ovary (right and left).
•	Numbers of the different ovarian follicle size categories (small: <3 mm, medium: 3–5 mm and large: >5 mm; Rodriguez et al. 2006).
•	Diameters, numbers and lifespan (duration of the functional corpus luteum, from the presence to the absence-regression – which corresponds to the luteal phase of the oestrous cycle, monitored by the laparoscope for a period of 18 d for the O + UT group and for 21 d for the O + FGA and I + FGA + eCG groups) of CL in each ovary.
•	Statistical analyses were carried out using Statview-IV programme (1996) on an IBM system. ANOVA repeated measure comparisons were used.

Results and discussion

Owing to the importance of sheep in general and the Awassi breed in particular, and since there is no information in the literature on the ovarian status of the Awassi ewe, there was a need to perform such a study. Laparoscopic examination was performed to assess the ovaries and the status of the ovarian response to the hormonal treatments inside and outside the breeding season. This reported work has helped to elucidate some other reproductive parameters in Syrian Awassi ewes as a continuation to previous studies (Zarkawi et al. 1999; Zarkawi 2000, 2004).

Progesterone concentrations as affected by the three experimental treatments

Progesterone concentrations have been extensively used in the studies of animal reproduction (Zarkawi 2007; Ai et al. 2009). They have been also used to confirm the clinical and morphological findings shown by laparoscopy (Caraty et al. 2007; Zarkawi and Soukouti 2010). Figure 1 shows mean serum progesterone concentrations in the blood of Syrian Awassi ewes in the three experimental groups during the study period. It can be observed that serum progesterone concentrations in the O + UT group were constantly very low (<2 nmol L⁻¹), as there were no CL and ewes were in an anoestrous period. However, cyclic patterns in serum progesterone concentrations were observed in ewes in the other two groups, indicating the presence of functional CL during the normal oestrous cycle (Zarkawi 1997).

Figure 1.  Means (±SE) of serum progesterone concentrations in the blood of Syrian Awassi ewes in the O + UT, O + FGA and I + FGA + eCG groups following laparoscopic measurements (three times a wk for a period of 18 d for the O + UT group and for 21 d for the O + FGA and I + FGA + eCG groups).

The ovaries

As shown in Table 1, ovaries of ewes in the O + UT group were very small, with an overall mean diameter of 1.24 cm, as ewes were in an anoestrous period. There were significant differences (p<0.05) in the diameters among ovaries in ewes in the O + UT group and in both O + FGA and I + FGA + eCG groups, but the difference in the diameter between the latter two groups was not significant (p>0.05).

Table 1. Mean diameters (±SD) of the right and left ovaries and the overall diameter (cm) in Syrian Awassi ewes in the O + UT, O + FGA and I + FGA + eCG groups following laparoscopic measurements (three times a wk for a period of 18 d for the O + UT group and for 21 d for the O + FGA and I + FGA + eCG groups).

Group	Right ovary diameter (cm)	Left ovary diameter (cm)	Overall diameter (cm)	Range (cm)
O + UT	1.24±0.07^aA	1.23±0.91^aA	1.24±0.08^aA	1.1–1.5
O + FGA	1.49±0.20^bB	1.44±0.14^bB	1.46±0.17^bB	1.2–2.0
I + FGA + eCG	1.55±0.13^cB	1.54±0.10^cB	1.55±0.12^cB	1.3–2.0
^a,bMeans in the same row with different superscripts indicate a significant difference (p<0.05).
A, B Means in the same column with different letters indicate a significant difference (p<0.05).

Within the same group, there were no significant differences (p<0.05) in the diameters between the right and left ovaries in the three groups.

The ovarian follicles

Numbers of the different ovarian follicles in the ovaries of three groups of Syrian Awassi ewes during the study period are shown in Table 2. Results indicated that <3 mm and 3–5 mm follicles were the least in number, and unlike the other two groups, there were no follicles larger than 4 mm found in the ovaries of ewes in the O + UT group; and follicles sized 3–4 mm were found only in 15 out of 98 ovaries as ewes were in an anoestrous period. However, follicles larger than 5 mm in diameter were found only in the ovaries of the I + FGA + eCG group (five 6-mm-sized follicles were observed in three ovaries). Saloia ewes treated with eCG had a higher number of follicles over 5 mm in diameter in the ovaries than those untreated (Silva et al. 2003). ECG has been used to improve ovulation rate (Lamrani et al. 2008) and, consequently, fecundity rate (Husein et al. 2007).

Table 2. Mean numbers (±SD) of the different ovarian follicles (<3 mm, 3–5 mm and >5 mm) and their ranges in the ovaries of Syrian Awassi ewes in the O + UT, O + FGA and I + FGA + eCG groups following laparoscopic measurements (three times a wk for a period of 18 d for the O + UT group and for 21 d for the O + FGA and I + FGA + eCG groups).

	Group
Follicle diameter category	O + UT Mean±SD* (range)	O + FGA Mean±SD (range)	I + FGA + eCG Mean±SD (range)
<3 mm	5.8±1.7^a (3–11)	10.6±1.8^b (7–15)	9.9 ±2.4^b (7–15)
3–5 mm	0.37±0.64^a (0–2)	1.4±1.0^b (0–4)	1.9±1.5^b (0–7)
>5 mm	0	0	(0–3)
*In both ovaries.
^a,bMeans in the same row with different superscripts indicate a significant difference (p<0.05).

There were no significant differences (p>0.05) in the numbers of follicles in each size category between the right and left ovaries within the same group. Using a laparoscope, similar results were reported by Kaulfuss et al. (1994) in Merino sheep.

The corpora lutea

In the present study, CL in the ovaries of ewes in the O + FGA and I + FGA + eCG groups developed normally from around day 4 of the oestrous cycle; a similar result was reported by Oldham and Lindsay (1980). However, CL started regressing between day 12 and day 15 of the oestrous cycle. Similar results were reported by Wani and Sahni (1988) in Muzaffarnagri ewes. The findings of the present study confirm earlier results reported by Zarkawi (2004) who, based on progesterone concentrations, reported the different phases of the oestrous cycle in hormonally-synchronised Syrian Awassi ewes as an Early luteal phase (2–5 d), a Luteal phase (8–12 d) and a Follicular phase (2–5 d).

No CL were found in the ovaries of ewes in the O + UT group, as ewes were in an anoestrous period. However, functional CL were formed in the ovaries of ewes in the O + FGA and I + FGA + eCG groups which were significantly higher (p<0.05) in the latter group in terms of numbers, diameters and lifespan (Table 3). Likewise, the mean progesterone concentration during the luteal phase was significantly higher (p<0.05) in the latter than in the former group (Table 3).

Table 3. Numbers, diameters and lifespans of corpora lutea (CL), and progesterone concentrations (nmol L⁻¹) (mean±SD) and their ranges in Syrian Awassi ewes in the O + UT, O + FGA and I + FGA + eCG groups following laparoscopic measurements (three times a wk for a period of 18 d for the O + UT group and for 21 d for the O + FGA and I + FGA + eCG groups).

Group	Number of functional CL* (range)	Diameter (mm) (range)	Lifespan (d) (range)	Progesterone (nmol L^−1) (range)
O + UT	None	–	–	0.61±0.58^c (0.01–1.96)
O + FGA	1.6±0.5^a (1–2)	8.1±1.3^a (5–10)	10.7±1.1^a (10–13)	10.23±5.28^a (3.28–23.81)
I + FGA + eCG	2.4±0.5^b (2–3)	8.8±1.8^b (5–15)	13.0±2.1^b (10–15)	20.79±14.31^b (3.29–65.98)
*In both ovaries.
^a,b,cMeans in the same column with different superscripts indicate a significant difference (p<0.05).

In the present study, no signs of adhesion formation were observed among ewes in the studied organs following repeated laparoscopic observations. Similar results were reported by Oldham and Lindsay (1980). Moreover, none of the ewes suffered complications or died during the course of examinations as a result of repeated laparoscopy; consequently, the mortality rate among all ewes was 0% throughout the study period. Similar results were reported in Damascus does (Zarkawi and Soukouti 2010). Therfore, laparoscopy is considered as a simple, accurate and practical technique for visual examionaion of abdominal organs (Maiti et al. 2008).

Conclusions

It might be concluded that laparoscopy could be safely, successfully and repeatedly applied on the same fat-tailed Syrian Awassi ewe without health problems and that the technique employed could also be useful in future AI programmes aiming at improving the reproductive efficiency of small ruminants in Syria.

Acknowledgements

The authors would like to thank the Director General and the Head of the Department of Agriculture of the Atomic Energy Commission of Syria for their support.