Elsevier

Theriogenology

Volume 71, Issue 8, May 2009, Pages 1267-1275
Theriogenology

Effect of type of semen, time of insemination relative to ovulation and embryo transfer on early equine embryonic vesicle growth as determined by ultrasound

https://doi.org/10.1016/j.theriogenology.2008.12.020Get rights and content

Abstract

Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm previous results in different settings and characterise the causes for this delay in growth. A total of 159 pregnancies from different mating protocols: (1) pre-ovulation natural mating, (2) pre-ovulation natural mating and transfer into recipient mares, (3) post-ovulation natural mating, and (4) post-ovulation AI with frozen/thaw spermatozoa were evaluated ultrasonographically from day 12 to 19 of pregnancy and vesicle diameters recorded. Regression analysis between embryonic vesicle diameters and embryonic ages was performed for each group and mean vesicle diameter at different age periods among groups were tested for statistical difference with a general linear model of variance. There was no significant difference between groups 1 and 2 (P = 0.73) or between groups 3 and 4 (P = 0.71). However both pre-ovulation groups (1 and 2) had larger vesicle diameters (P < 0.000) at any embryonic age analysed than either of the post-ovulation groups (3 and 4). In conclusion, post-ovulation inseminations produced pregnancies with smaller vesicle diameters equivalent to approximately 1 day's growth.

Introduction

The horse embryo is conceived in the ampulla, near the junction with the isthmus where is retained until the beginning of oviductal descent approximately 4 days post-fertilization [1] and culminates when the embryo enters the uterus sometime between 6 and 6.5 days after ovulation [2], [3]. During the oviductal stage, the embryo does not increase in size remaining not different from the original unfertilized oocyte with a diameter range from 149 to 178 μm [2]. At the uterine stage the conceptus begins a rapid growth phase in which the vesicle diameter increases gradually maintaining its spherical shape until about 17–18 days post-ovulation when it reaches a plateau and the vesicle outline becomes irregular as it intimates with the endometrial folds.

Monitoring of conceptus diameter is easily and routinely performed in practice by transrectal ultrasonography. Although the minimum vesicle size visible with the scanner would depend on the probe resolution and experience of the operator, in most occasions vesicles of 3–4 mm can be accurately identified. The mean day of first detection of embryonic vesicles has been reported to be 11.1 days post-ovulation with a mean vesicle diameter of 6.7 mm [4]. Earlier assessment of vesicle diameter is also possible by measurement of embryos recovered by uterine flushing at known stages of development. Several factors affecting the early embryonic vesicle diameter have been proposed: time of insemination relative to ovulation [4], number and synchrony of multiple ovulations [5], presence of multiple vesicles in the uterus [5], [6], [7], degree of synchrony between donor and recipient mares in embryo transfer technology [8], health of uterus (Newcombe and Cuervo-Arango, unpublished) and age of mare and oocyte quality [9].

Knowledge of embryonic vesicle diameter in relation to ovulation date during earlier stages and the shape and position of the embryo proper within the conceptus thereafter may have clinical relevance for the practitioner since impending embryonic failure can be suspected in small for age vesicles [7] and in conceptuses with abnormal development of the embryo proper [10].

Embryonic age is either determined from the point in which ovulation is first detected (in pre-ovulation inseminations) or from the point of insemination (in post-ovulation inseminations). Accuracy of determining the embryonic age will depend on the frequency of ultrasound examinations to detect ovulation (pre-ovulation inseminations) or on the assumption that fertilization occurs soon after insemination (post-ovulation inseminations).

In pre-ovulation inseminations equine sperm is known to reach the oviducts as early as 0.5 h post-insemination [11], however it has been proven that sperm located in the uterine lumen between 2.5 and 4 h post-insemination is still involved in fertilization since uterine lavage with large volume of saline 2.5 h but not 4 h after insemination, decreased pregnancy rates significantly [12], [13]. The time taken for the oocyte to reach the fertilization site is assumed to be short like in other domestic species such as the pig in which the oocytes take about 30–45 min [14], [15]. Therefore it could be assumed that fertilization after post-ovulation insemination would occur somewhere at or soon after 4 h post-insemination and yet difference in vesicle diameter equivalent to 1 day's growth was found between mares inseminated before and after ovulation [4]. In the latter study however, embryonic ages of the pre-ovulation group were known only to with ±12 h accuracy. The authors concluded that the difference in growth could be attributed, at least in part, to requirements for sperm capacitation in the post-ovulation group. If the cause for this delay were the requirements for sperm capacitation, it could be hypothesised that post-ovulation insemination with frozen semen (which after thawing acquires a capacitation-like status [16]), would fertilize the oocyte faster than fresh spermatozoa.

The objectives of this study were to determine (a) the effect of different types of semen (frozen and fresh) on the time taken from insemination to fertilization and subsequent embryonic development and (b) the effect of embryo transfer on early embryonic vesicle development. It was hypothesised that fertilization after insemination with frozen semen would occur faster than that with fresh semen in post-ovulation inseminations and that the procedure of embryo handling and asynchrony of between donor and recipient mares would retard post-transfer embryonic vesicle growth.

Section snippets

Animals and ultrasound measurements

Embryonic vesicle diameters in mares of various breeds (Irish draught, Warmblood and Thoroughbred) resident in a fertility veterinary clinic were evaluated by ultrasonography with a 7.5 MHz linear probe during the 2007 and 2008 breeding seasons. The vesicle diameter was obtained from average of two linear measurements of the conceptus taken at right angles when the image of the vesicle was maximum using the electronic callipers.

Experimental design

Different mating protocols were evaluated to study the relationship

Results

Regression curves of embryonic vesicle diameters of groups 1 and 2 as well as of groups 3 and 4 did not visibly differ respectively during the recorded period as shown in Fig. 1. Regression fits of vesicle diameters from pooled data of pre-ovulation and post-ovulation groups showed however a clear discrepancy in size equivalent to approximately 1 day's growth (Fig. 2). Vesicle diameters of both pre-ovulation insemination groups were not significantly different (P = 0.73). In the same way,

Discussion

This study focused on the effect of time of insemination relative to ovulation, type of semen used and effect of embryo handling and transfer on early embryonic vesicle diameter and growth measured by ultrasonographic examination. The results showed that the only factor affecting the vesicle diameter at a particular known age of pregnancy was whether spermatozoa were present in the oviducts at the time of ovulation. Neither embryo transfer nor type of semen (fresh versus frozen) had a

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