Development of in vitro embryo production systems for red deer (Cervus elaphus)

Animal Reproduction Science 70 (2002) 65–76

Development of in vitro embryo production systems for red deer (Cervus elaphus) Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes D.K. Berg a,c,∗ , J.G. Thompson a,1 , G.W. Asher b a

Reproductive Technologies Group, AgResearch Ruakura, Private Bag 3123, Hamilton, New Zealand b AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel, New Zealand c Department of Animal Science, University of Minnesota, St. Paul, MN, USA

Received 30 January 2001; received in revised form 29 November 2001; accepted 13 December 2001

Abstract In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4–12 h on COEM or for 4 h in TALP medium supplemented with 20 ␮g of heparin. COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm (P < 0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24 h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4 h and no motile sperm were observed by 8 h. Overall, the penetration rates of in vitro red deer oocytes were low (5–28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin (P < 0.001). Penetration rates were similar for 4–12 h pre-incubation of sperm on COEM (P > 0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24 h compared to 12 h (P < 0.001). There were no differences in penetration rates among the four donor stags used in the study. ∗ Corresponding author. Tel.: +64-7-838-5538; fax: +64-7-838-5536. E-mail address: [email protected] (D.K. Berg). 1 Present address: The University Department of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, Woodville Road, Woodville, SA 5011, Australia.

0378-4320/02/$ – see front matter © 2002 Published by Elsevier Science B.V. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 9 9 - 3

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It was concluded that COEM sustains red deer sperm motility in vitro during the 24 h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used. © 2002 Published by Elsevier Science B.V. Keywords: Red deer; Cervus elaphus; In vitro fertilisation; Oviduct epithelial cells; Heparin

1. Introduction In vitro produced embryo (IVP) technology plays the same role for farmed cervids as for traditionally farmed species, namely, the production of large quantities of embryos for transfer and oocytes/embryos for other manipulations, such as embryo sexing, gene injection and cloning. The establishment of in vitro technology would also enhance the creation of new hybrid species that would not normally occur, add to the knowledge of comparative biology and may assist in captive breeding programs for endangered cervid species. IVP has been successfully applied to cattle, sheep and goats, with transferred embryos resulting in live offspring (Gordon, 1994). However, results have been disappointing when bovine in vitro embryo production systems (with or without co-culture techniques) have been applied to the cervid species (red deer, Fukui et al., 1991; Bainbridge et al., 1999; reindeer, Krogenaes et al., 1994; wapiti, Pollard et al., 1995; axis deer, Chapman et al., 1999). In vitro maturation (IVM) rates of oocytes using either M199 or Hams F-10 supplemented with hormones and foetal or steer serum were acceptable in all studies. However, in vitro fertilisation (IVF) comparisons among these are difficult to interpret due to species variation, regional seasonal differences and type of sperm used for IVF (i.e. frozen-thawed versus fresh and electroejaculated versus epididymal). Red deer and reindeer IVF rates were poor, with few oocytes cleaving and no development beyond the eight-cell stage (Fukui et al., 1991; Bainbridge et al., 1999; Krogenaes et al., 1994). In contrast, a 31% blastocyst rate was reported when red deer and wapiti oocytes were co-incubated with wapiti sperm in the presence of bovine oviductal explants (Pollard et al., 1995). Thus, apparent species differences occur for in vitro fertilisation conditions, even between taxa (subspecies) as closely related as the red deer and wapiti. A previous red deer study used semen from one stag (Fukui et al., 1991). It is well established that semen from individual bulls and rams exhibits a high degree of variability during in vitro capacitation and fertilisation (Fukui et al., 1988; Hillery et al., 1990; Holm et al., 1994). Therefore, it is questionable whether this initial result in red deer reflected the inability of the IVF system to support red deer fertilisation in general or simply the use of a stag not suitable for IVF. Furthermore, fertilisation failure does not necessarily imply capacitation failure, as successful IVF involves a series of steps, each of which needs to be performed in strict sequence. The first step requires a population of motile sperm to reach and penetrate the egg investments and bind to the zona pellucida (Yanagimachi, 1994). Preliminary studies have shown that red deer sperm lose their motility within 4 h of in vitro incubation when several different ruminant fertilisation formulations were tested (Berg, 1997).

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Recent advances in tissue culture techniques have led to the culture of oviduct epithelial cells from a variety of mammalian species, and an in vitro model for co-culture of sperm with either suspensions or monolayers of oviduct epithelial cells has been reported (Nagai and Moor, 1990; Pollard et al., 1991; Suarez et al., 1991; Gutierrez et al., 1993). In these systems, sperm rapidly attach to the cultured oviduct epithelial cells and are slowly released over a period of days. Pollard et al. (1991) demonstrated that the motility and fertilisability of frozen-thawed bovine sperm could be maintained for up to 36 h when the sperm were bound to oviduct epithelial cells. The motility of stallion sperm was maintained for 4 days when the sperm were bound to the epithelial cells compared with 12 h when they were incubated in medium alone (Ellington et al., 1993b). This ability of oviduct epithelial cells to retain sperm motility and viability has been demonstrated in cattle (Guyader and Chupin, 1991; Pollard et al., 1991); goats (Barahona et al., 1997); sheep (Gutierrez et al., 1993) and horses (Ellington et al., 1993a,b). Furthermore, conditioned medium (Cm) from the oviduct epithelial cells has also been shown to maintain the motility of bovine and ovine spermatozoa (Gutierrez et al., 1993; Ijaz et al., 1994). This was the first in a series of studies designed to develop an in vitro embryo production system for New Zealand farmed red deer (Cervus elaphus scoticus). The ultimate purpose is to produce calves from high genetic value females using assisted reproductive technologies. The objective of the present study was to determine the requirements for IVF with red deer spermatozoa. Experiment 1 compared the motility of red deer sperm when incubated on red deer oviduct epithelial cell monolayers (COEM), Cm or medium alone. Experiment 2 investigated the ability of COEM to capacitate red deer sperm as determined by the ability to penetrate red deer IVM oocytes.

2. Materials and methods All chemicals were BDH Analar grade unless otherwise stated. 2.1. Preparation of oviduct epithelial monolayers In each replicate, oviducts were selected from a pool of randomly cycling hinds (N = 96) slaughtered at a venison processing plant during the month of April. One oviduct (ipsilateral to the preovulatory follicle) from two separate hinds was used for each replicate. The criteria used to select a preovulatory oviduct were based upon the observations of surgical recovery of ova from synchronised hinds (0–36 h after the LH surge) in a previous experiment (Berg, 1997). A follicle larger than 7 mm in diameter was present in conjunction with a regressing CL, which was pale pink to white in colour, and the uterine horns had marked tonus and were reddish in colour. At collection, the oviducts were placed in H199 supplemented with 10% foetal calf serum and held at 25 ◦ C (2 h) until processing. Epithelium was extruded as a tube by passing a sterile glass slide along the outside of the oviduct, from the uterotubal junction to the infundibulum. The extrusion, containing both ampullar and isthmic cells, was disaggregated by repeated passages through a 25-gauge needle. The cells were washed twice by consecutive suspensions and centrifugations (500 g for 5 min) in Hepes buffered (20 mM) TCM 199 (H199, pH 7.4, Earle’s salts with l-glutamine and

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without sodium bicarbonate; Gibco Laboratories Inc., Grand Island, New York, USA) supplemented with 25 ␮g kanamycin sulfate (Sigma, St. Louis, MO, USA), 4 mM NaHCO3 , 0.33 mM pyruvate and 10% heat inactivated deer serum (DS, collected 24 h after oestrus). The cells were resuspended in B199 (TCM 199, pH 7.4, Earle’s salts with l-glutamine and without sodium bicarbonate; Gibco Laboratories Inc., Grand Island, New York, USA) supplemented with 25 ␮g kanamycin sulfate (Sigma, St. Louis, MO, USA), 25 mM NaHCO3 , and 0.33 mM pyruvate supplemented with 10% DS and were allowed to settle for 15 min at 39 ◦ C in humidified 5% CO2 in air. A sample (50 ␮l) was placed on a warmed glass slide and observed at 200× magnification using phase-contrast microscopy. Oviducts were considered suitable for use if the ciliated cells were vigorously beating. Aliquots (50 ␮l) of this suspension were transferred to 450 ␮l of B199 in individual wells of a 24-well flat-bottomed plastic tissue culture plate (Falcon Plastics, Los Angeles, CA). Cells were cultured at 39 ◦ C under a humidified atmosphere of 5% CO2 in air. Medium was replaced every second day and confluent monolayers were formed in 7 days. The growth medium was replaced with Sp-TALP medium (Parrish et al., 1988) 12 h before the addition of sperm. The epithelial cell cultures were rinsed once and re-equilibrated with Sp-TALP. Conditioned Sp-TALP medium (Cm) was obtained by incubating TALP with oviduct epithelial monolayers for 12 h. Cm was harvested by aspirating the Sp-TALP medium off three or four individual wells and pooled together before centrifugation. The medium was centrifuged for 5 min at 500×g to remove any cellular debris. The supernatant was collected and 500 ␮l aliquants were immediately placed into individual wells of flat-bottomed 24-well plates. The wells were placed back into an incubator to equilibrate (2 h) before adding the sperm. A new batch of Cm was made for each replicate (N = 6). Samples of the confluent oviduct epithelial cell monolayer with attached sperm were stained with the supervital stain Hoechst 33342 (l ␮g ml−1 ) for 30 min before viewing under an epifluorescence microscope (Nikon Diaphot) to assess viability of the oviduct and sperm cells. The samples were viewed after 12 h of incubation. 2.2. Experiment 1: the effect of red deer oviduct epithelial cell monolayers on the maintenance of sperm motility 2.2.1. Preparation of sperm Red deer sperm motilities over time were compared when incubating sperm in Sp-TALP alone, Cm and COEM. Frozen-thawed semen from two fertile stags was used. A limited number of straws from each stag were available for these experiments so the semen was pooled for the motility experiments. Electroejaculation, semen processing and freezing was done as described by Asher et al. (1988). Two straws from each of two red deer stags were thawed, pooled and washed twice by consecutive suspensions and centrifugations (300×g for 5 min) in Hepes buffered Sp-TALP. The sperm pellet was divided equally into three conical centrifuge tubes and adjusted to a final volume of 1 × 106 sperm ml−1 with three different media treatments: (1) Sp-TALP; (2) Cm; (3) transfer of sperm directly onto the epithelial cell cultures. Motility was visually assessed using phase-contrast optics (200×) for treatments 1 and 2. Sperm motility on the epithelial oviduct monolayers was assessed as described by Pollard et al. (1991). Briefly, the percentage of bound sperm was estimated by averaging the percentage from three fields

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of view. One hundred bound sperm were examined using an inverted microscope fitted with phase-contrast optics (200×) and were classified as motile or non-motile (at 0, 4, 8, 12 and 24 h). Sperm with tail movement were judged as motile. 2.3. Experiment 2: the effect of red deer oviduct epithelial monolayers on the fertilising capacity of red deer sperm 2.3.1. Preparation of sperm The ability of red deer sperm to penetrate in vitro mature red deer oocytes that were pre-incubated on COEM for 4 or 12 h was compared with incubation with heparin alone. Frozen-thawed sperm from four different red deer stags were used throughout the experiment. Three sperm treatments were investigated: (1) 4 h pre-incubation in Sp-TALP + 20 ␮g heparin; (2) 4 h incubation on the oviduct monolayer; (3) 12 h incubation on the oviduct monolayer. Monolayers were prepared as described for experiment 1. The swim-up method (Parrish et al., 1986) was used to collect the greatest percentage of motile sperm. For each individual stag, two straws were thawed at 35 ◦ C for 30 s and the contents (450 ␮l) were overlaid with 1.5 ml of Sp-TALP and maintained at 39 ◦ C for 1 h. The top 1 ml was taken and motility and concentration determined for each individual stag. Sperm motility was consistently found to be 60–75% throughout the experiment. The motile fraction was then pelleted by centrifugation at 500 × g to concentrate the sperm and then resuspended in Sp-TALP at a concentration of 10 million sperm ml−1 . A 50 ␮l aliquant of the sperm suspension was placed on the monolayer containing 450 ␮l of Sp-TALP. The final sperm concentration was 1 × 106 sperm ml−1 . The sperm were incubated upon the monolayer for 4 or 12 h at 39 ◦ C in an atmosphere of 5% CO2 in air. Before the addition of oocytes, half (250 ␮l) of the Sp-TALP was replaced with 250 ␮l of Fert-TALP (Parrish et al., 1988). In addition, a heparin control was included. For this, 10 million sperm ml−1 were incubated for 4 h in Sp-TALP medium containing 20 ␮g of heparin (Sigma, St. Louis, MO, USA) in a flat bottom well of individual 24-well plates. Prior to the addition of oocytes, 50 ␮l of the sperm suspension was added to a well containing 450 ␮l of Sp-TALP. After 10 min of equilibration, half (250 ␮l) of the Sp-TALP was replaced with 250 ␮l of Fert-TALP. A 12 h pre-incubation time in Sp-TALP alone was not included because the motility of the sperm was less than 10% by this time. 2.3.2. Oocyte maturation Ovaries (approximately 80 for each replicate) from randomly cycling red deer were obtained at the time of slaughter from a venison processing plant during the breeding season (April through June). Ovaries were collected 15 min after slaughter; placed into warm saline and maintained at 30 ◦ C during the 2.5 h collection period and the 1 h transport time back to the laboratory. Oocytes were aspirated from 2 to 6 mm follicles using a 20-gauge needle under vacuum pressure (32 mm Hg) into a 15 ml test tube containing aspiration medium (H199 supplemented with BSA (0.4% liquid BSA, Immuno Chemical Products, Auckland, New Zealand), 20 mM Hepes, 4 mM NaHCO3 . Oocyte processing and selection took 1.5 h. The 7–10 cumulus enclosed oocytes were placed into in 50 ␮l of maturation medium (B199 supplemented with 10 ␮g of LH and FSH (Immuno Chemical Products,

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Auckland New Zealand), 1 ␮g of estradiol (St. Louis, MO, USA) and 10% fetal bovine serum (Gibco Laboratories Inc., Grand Island, New York, USA) under mineral oil (Squibb, Princeton, NJ, USA) 2.5–5 h after slaughter. Oocytes were incubated at 39 ◦ C in a humidified atmosphere consisting of 5% CO2 in air for 24 h. At that time, 10–15 oocytes were fixed in ethanol:acetic acid (3:1 v/v) for 48 h at room temperature (20–25 ◦ C), stained with lacmoid (1%) and evaluated for nuclear maturation. 2.3.3. In vitro fertilisation IVM oocytes were manually removed from the expanded cumuli oophori using a 5 min incubation in 0.1% hyaluronidase (St. Louis, MO, USA) in H-TALP (Parrish et al., 1988) while gently pipetting the oocytes through a finely pulled pipette attached to a 1 ml syringe. Approximately 25% of the zona pellucida was exposed. Oocytes (20–25) were added to the sperm treatments and gametes were co-incubated for either 12 or 24 h at 39 ◦ C in 5% CO2 in air. At the end of co-incubation, oocytes were fixed and stained as described and then examined by phase-contrast microscopy (400× magnification) to determine rates of sperm penetration and polyspermic penetration. Sperm penetration was defined as the presence of a whole sperm, a sperm head, or a male pronucleus with associated sperm tail in the ooplasm. Polyspermic penetration was defined as the presence of any multiple of sperm structures within a single oocyte. Any oocyte in which these structures could not be clearly seen was categorised as unfertilised. 2.4. Statistical analyses Motility data were transformed for each time point using the angular (arcsine) transformation. A randomised block design with day (replicate) as a block and media and time as the treatments were analysed by Analysis of Variance using Genstat 4.1 for Windows (Lawes Agricultural Trust, UK). Individual means among media were compared using Fisher’s least significant difference. Penetration rate data (logit transformed) were analysed by a balanced least-squares analysis of variance using the Genstat statistical package for binomial data. Factors tested included stag, replicates, length of co-incubation time, and time of sperm incubation. 3. Results 3.1. Experiment 1 Co-incubation of red deer sperm with COEM was superior in maintaining sperm motility over the 24 h period (P < 0.05). Motility was sustained at levels comparable to the initial motility rating over the 24 h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4 h and no motile sperm was observed by 8 h (Fig. 1). Sperm had bound to the COEM within 1 h of incubation. The density of attachment was not uniform across the monolayer. Sperm were closely spaced in clusters in some areas,

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Fig. 1. Changes in mean (±S.E.M.) red deer sperm motility over a 24 h period incubated in Sp-Talp (䉲); conditioned medium (䊊); and cervine oviduct epithelial monolayer (䊉).

sparsely in others, and a few areas had no sperm attachment (Fig. 2). By 12 h, some of the sperm had begun to detach and an increase in free-swimming sperm was observed. Hyperactivity was observed in a small percentage, estimated to be up to 10% of the free-swimming sperm, but was not detected in every replicate (N = 6). The majority (estimated to be 90–95%) of the detached sperm were non-motile by 24 h. 3.2. Experiment 2 All sperm samples collected after the swim-up were 60–75% motile. There were no differences in sperm motility among the four stags. Oocyte maturation (mean ± S.E.M.) was 69.4 ± 5.4%. Table 1 Penetration of IVM red deer oocytes by red deer sperm incubated with or without oviduct epithelial monolayers Treatment

12 h

Total % ± S.E.M.

24 h

n

(%)

n

(%)

Sp-TALP + (4 h) heparin Monolayer (4 h) Monolayer (12 h)

145 150 133

5.5 10.0 10.5

112 112 120

10.7 20.5 28.0

Total

428

344

17.9 ±1.8 B

8.3±1.2 A

7.2 ± 1.5 a 15.1 ± 2.1 b 17.2 ± 2.1 b

(A, B) P < 0.001 denotes difference between 12 and 24 h; (a, b) P < 0.001 denotes differences within columns.

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Fig. 2. Attachment of red deer sperm on homologous oviduct monolayers. Sperm incubated for 12 h. (a) Differential interference contrast microscopy using both bright light and fluorescence together. Sperm attachment is in large or small clusters and with no attachment in other areas. (b) Higher magnification showing sperm attachment to a secretory-type epithelial cell. Both light and fluorescence microscopy. (c) Fluorescence only of (b). Bright fluorescence is the sperm nuclei. Less intense fluorescence is the epithelial cell nuclei.

Fertilisation rates overall were low across different media (5–28%, Table 1). However, sperm pre-incubated on oviduct monolayers penetrated more oocytes than sperm incubated with heparin (P < 0.001). Fertilisation rates were greater across all treatments when both sperm and oocytes were co-incubated for 24 h compared to 12 h (P < 0.001). Fertilisation rates were similar for 4 and 12 h pre-incubation of sperm on the oviduct monolayers (P > 0.50). There were no differences in fertilisation rates among the four stags. However,

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a wide range of penetration rates for each stag was observed, which was reflected in a significant replicate effect for sperm penetration (P < 0.001). 4. Discussion The results of experiment 1 have demonstrated that incubating red deer sperm on oviductal monolayers maintained red deer sperm motility up to 24 h. Similarly, contact with oviductal epithelium has been shown to be beneficial for sperm motility in vitro (Pollard et al., 1991; Suarez et al., 1991; Ellington et al., 1993a) and in vivo (Smith and Yanagimachi, 1990, 1991) for several species. The mechanism by which cultured cells exert their effect is unknown, although it is thought that motility factors may be adsorbed onto the spermatozoa when they come into contact (bind) with the cultured cells or they may be secreted into the medium. Also, toxic products of sperm metabolism may be neutralised by the cultured cells to help maintain motility (Fujihara and Howarth, 1980; Ashizawa et al., 1986). Sperm incubated in both conditioned medium from red deer epithelial cell monolayers and TALP medium alone had similar motility and were of lower motility than those incubated on oviductal cells. This result differs from bovine and ovine data which demonstrated that conditioned medium harvested from oviductal epithelial cells prolonged sperm motility compared with medium alone (Gutierrez et al., 1993; Ijaz et al., 1994). These authors suggested that a motility factor produced by the oviduct epithelial cells was secreted into the medium and that this factor maintained sperm motility when added back to Sp-TALP medium (Ijaz et al., 1994). Abe et al. (1995) have isolated and purified a bovine oviductal glycoprotein from epithelial cell cultures. This glycoprotein maintains sperm motility in a dose dependent manner and is associated with the posterior head, middle portion and tail of bovine spermatozoa. One difference between this study and that of previous studies may be due to the length of time the medium was “conditioned” before harvesting. Red deer conditioned medium was used after 12 h conditioning on epithelial cells, whereas bovine medium was conditioned for 2–5 days (Gutierrez et al., 1993; Ijaz et al., 1994; Abe et al., 1995). The 12 h conditioning time may not have been enough time for the motility factor(s) to be secreted into the fresh medium at an effective concentration to maintain sperm motility. In experiment 2, the capacity of homologous epithelial oviduct monolayers to induce red deer sperm capacitation was assessed by the ability of these sperm to penetrate IVM red deer oocytes. The fertilisation rate doubled when sperm were pre-incubated on oviduct monolayers rather than sperm capacitated with heparin alone. This contrasts to bovine and caprine data where little or no improvement in fertilisation has been reported when sperm are incubated in the presence of oviduct epithelial cells (Choi et al., 1991; Pollard et al., 1991; Holm et al., 1994; Chian and Sirard, 1995; Barahona et al., 1997). However, these reported fertilisation rates were considerably higher (70–95%) using either chemical capacitation or oviduct epithelial cells than found for any media used for red deer. It has been shown that a heparin-like glycosaminoglycan from the bovine oviduct is a potential in vivo capacitation agent (Parrish et al., 1989). In addition, a factor present in bovine oviduct conditioned medium was shown to have similar activity to the heparin-like glycosaminoglycan from the bovine oviduct during in vitro fertilisation (Chian and Sirard,

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1995). Two reasons may explain the low fertilisation rate for red deer sperm incubated on cervine oviduct epithelial monolayers. Either, in vitro cultured red deer oviduct epithelial cells did not secrete a capacitation factor under the in vitro conditions reported here, or heparin or a heparin-like glycosaminoglycan is not an effective capacitation agent for red deer sperm. The latter is supported by the low fertilisation rates observed in the present study (5–11%) and by Fukui et al. (1991; 5–24%) when heparin was the capacitating agent. The fertilisation rate doubled when gametes were co-incubated for 24 h, regardless of capacitation treatment. The highest penetration rate (28.0%) was achieved when sperm were pre-incubated for 12 h and co-incubated for 24 h. The majority (80%) of stained oocytes had formed male pronuclei after 24 h of co-incubation. Assuming that the time required for red deer to form pronuclei is similar to that of the bovine (12–14 h) (Xu and Greve, 1988), the majority of the oocytes were not penetrated until they had been in fertilisation medium 10–12 h. This suggests that red deer sperm required either 16–24 h pre-incubation before penetrating the oocytes. Fertilisation results may have improved if sperm had been pre-incubated for 18–24 h rather than the 12 h period. There was a slight increase in sperm motility after 12 h incubation on the oviduct monolayers. This was interpreted to indicate that the red deer sperm were beginning to be released from the epithelial monolayer and a portion of the sperm may have begun to capacitate. It has been speculated that capacitation is required for sperm to release from the oviduct epithelium. Results have shown that in vitro capacitated hamster sperm do not bind to in vivo oviduct epithelium (Smith and Yanagimachi, 1991); while in vitro capacitated bovine sperm show reduced binding to oviduct epithelial explants (Lefebvre and Suarez, 1996). Unexpectedly, no difference was observed in the ability of sperm from different stags to penetrate IVM red deer oocytes. It may be that sperm penetration was not sensitive enough to detect fertilisation differences in the stags because the penetration rates were low (5–28%). Overall, the fertilisation rates were comparable to those reported for red deer by Fukui et al. (1991; 5–24%;) even though differences in heparin concentration, fertilisation systems and stags were used. The use of homologous oviduct epithelial monolayers does increase the rate of in vitro fertilisation, but at a rate too low and variable to be used on a routine basis. Additionally, continuous collection of oviducts at the correct stage of the cycle becomes increasingly difficult because of the seasonality of the species. Furthermore, hinds within the same herd are usually at the same stage of their oestrous cycle or pregnancy at slaughter (D.K. Berg, personal observation). For oviductal monolayers to be of practical use in red deer in vitro embryo production, the use of frozen-thawed oviduct cells needs to be examined.

Acknowledgements This work was completed while the primary author was supported by a Fullbright Scholarship. The authors thank the Ruakura Deer Group for help in semen collection, F. Nemaia for processing and freezing of the semen and Summit Deer Products Ltd., for the supply of ovaries.

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