A Preliminary Report of Laparoscopic Oocyte Collection in Ewe Lambs and Aged Ewes

Treated with Follicle Stimulating Hormone During the Breeding Season;

J.S. Luther, D. Pant, J.T. Choi, C. Navanukraw, J. Beckman, J.D. Kirsch, R. M.Weigl, K.C.

Kraft, D.A. Redmer, L.P. Reynolds, and A.T. Grazul-Bilska

Department of Animal and Range Sciences, North Dakota State University, Fargo, ND, USA

INTRODUCTION

The potential application of in vitro embryo production (IVP) technologies partially depends on the development of reliable, repeatable and efficient techniques for recovery of oocytes from genetically valuable animals. While oocyte collection from living animals has been widely used in the bovine species, its exploitation in sheep has been limited (Galli et al., 2001). Most work reported in sheep has been accomplished with slaughterhouse-derived oocytes (Naitana et al., 1992; Guler et al., 2000; Gordon, 1997; Cognie, 1999).

However, Snyder and Dukelow (1974) pioneered laparoscopic oocyte collection in sheep, and this technique was further improved in recent years (Tervit et al., 1992; Baldassarre et al., 1994; Earl and Kotaras, 1994; Stangl et al., 1999). Laparoscopic oocyte collection is an effective and minimally invasive technique, which offers the possibility of repeated ovum pick-up and allows for repeated production of embryos from a single donor ewe. Nor does this technique cause permanent damage to the donor ewe's reproductive health. In addition, the donor ewe can be in almost any physiological status and still be suitable for oocyte recovery (Gordon, 1997).

The objective of this study was to determine if laparoscopic oocyte collection can be used as an efficient and effective technique for retrieving oocytes from ewe lambs and aged ewes treated with follicle stimulating hormone (FSH) during the breeding season.

MATERIALS AND METHODS

Animals, Experimental Design, and Oocyte Collection

Crossbred Columbia x Hampshire ewe lambs and Rambouillet x Targhee western range ewes aged (2-6years old) were used in this study (n=6/age group). All oocyte collections were performed over a period of three weeks during the normal breeding season (November and December, 2001). Only ewes having a normal estrous cycle (15-17 days; determined using vasectomised rams) were used. For induction of superovulation, the ewes were given intra-muscular (i.m.) injections of FSH-P (Sioux Biochemical, Sioux Center, IA) twice daily on days 13 and 14 of the estrous cycle before oocyte collection (Stenbak et al., 2001).

All ewes were kept off feed and water for a period of 24 h before oocyte collection. The collection was performed under sedation using Rompun (0.1 mg/kg i.m.) and Ketaset (1.25 μg/kg, i.m.; Gourley and Riese, 1990). Laparoscopy was performed as previously described Gourley and Riese, 1990). The position of the ovaries was viewed and the number of follicles on each ovary was counted. All visible follicles were aspirated with an 18-gauge double lumen ovum pick-up needle (Cook Veterinary Products, Spencer, Inc.) using oocyte collection media containing heparin (Stenbak et al., 2001).

After oocyte collection, the contents of the collection vial were transferred to a petri dish for observation under a stereomicroscope. Recovered oocytes were transferred into a petri dish with fresh collection medium without heparin (Stenbak et al., 2001) and the number of collected oocytes was determined.

Statistical Analysis

Numbers of follicles observed and aspirated, number of oocytes collected, and percentage of oocytes collected were analyzed by using the general linear models procedure of the Statistical Analysis System (User’s Guide, 1985).

RESULTS

Table 1 presents the number of follicles observed, number of follicles aspirated, number of oocytes collected and recovery rate of oocytes for ewe lambs and aged ewes.

Table 1. Number of observed follicles, aspirated follicles, oocytes collected and the percentage of oocytes collected for ewe lambs and aged ewes treated with FSH and subjected to laparoscopic oocyte collection.

Age group	Ear Tag #	No. of Follicles Observed	No. of Follicles Aspirated	No. of Oocytes Collected	Percentage of Oocytes Collected *
Lamb	114	18	4	2	50.0
Lamb	181	24	24	14	58.3
Lamb	20	16	16	7	43.8
Lamb	133	40	40	27	67.5
Lamb	15	37	31	15	48.4
Lamb	1455	27	22	7	31.8
Total	6	162	137	72	52.6
Mean+SEM/Ewe		27±4.0^a	22.8±5.1	12±3.6	52.6±5.0
Aged	10	14	14	4	28.6
Aged	93	16	16	4	25.0
Aged	24	9	9	4	44.4
Aged	34	14	15	3	20.0
Aged	62	30	30	11	36.7
Aged	87	24	22	17	77.3
Total	6	107	106	43	40.6
Mean+SEM /Ewe		17.8±3.1^b	17.7±3.0	7.2±2.3	40.6±8.5

*Percentage of oocytes collected = (no. of oocytes collected/no. of follicles aspirated) x 1000

^{a, b} values (means±SEM) are different; P<0.10.

Ewe lambs had more (P<0.10) visible follicles present on their ovaries than aged ewes. However, the number of follicles aspirated, the number of oocytes collected, and the percentage of oocytes collected were similar (P>0.10) for both the age groups.

DISCUSSION

Genetic improvement and livestock propagation by the conventional means of breeding is a slow process. For small ruminants, laparoscopic oocyte collection is the technique of choice for its simplicity, minimal invasiveness, repeatability and efficiency. Oocytes collected can be subsequently utilized for the in vitro production (IVP) of embryos (Earl and Kotaras, 1997). In addition, the rates of development are similar or even greater when the oocytes are recovered from live donors compared with oocytes collected from ovaries of slaughtered animals (Galli et al., 2001).

In the present experiment the recovery rate was 53% and 41% for lambs and aged ewes, respectively; other workers have shown similar or even higher results. A similar recovery rate of 49-54% was reported by Tervit et al.(1992) for ewes treated with PMSG. Stangl et al. (1999) also obtained a similar recovery rate (51 to 62%) when oocytes were collected once or twice a week from the same animal. In addition, Stangl et al. (1999) reported a greater recovery rate for ewes stimulated with PMSG compared with non-stimulated ewes, and also reported that 65-70% of the cumulus oocyte complexes (COC) were suitable for IVP of embryos. A greater rate of oocyte recovery ranging from 80 to 89% was reported by Baldassarre et al. (1994) and Earl et al.(1995) using the laparoscopic technique. These data demonstrate that the laparoscopic technique for oocyte collection is efficient and may provide large numbers of oocytes for IVP systems.

Data concerning the ability of oocytes collected via laparoscopy to fertilise in vitro are very limited at present. Only Tervit et al. (1995) reported the birth of lambs from embryos produced in vitro following laparoscopic recovery of oocytes. This indicates that this technique may find practical application for IVP of embryos. However, this subject requires further study.

In the present experiment the ewe lambs exhibited more visible follicles at collection compared with aged ewes, after FSH administration. Although the mechanisms responsible for this difference are currently unclear, it may result from differences in nutrition, breed, age or ability to respond to exogenous FSH administration. This unexplained difference represents another topic for future studies.

In summary, the present experiment demonstrates that laparoscopy is a feasible method of obtaining oocytes from donor ewes. The ewes lambs used in this study exhibited a better response to exogenous FSH administration and it seems that they may provide more oocytes for IVP of embryos. Future studies should confirm these age differences in FSH response, and also evaluate the quality of oocytes collected by laparoscopy by using in vitro fertilization procedures followed by embryo transfer.

ACKNOWLEDGMENTS

Supported in part by Hatch Project ND 01705 and ND SBARE.

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