Effect of type of semen, time of insemination relative to ovulation and embryo transfer on early equine embryonic vesicle growth as determined by ultrasound
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between donor and recipient mares in embryo transfer technology [8], health of uterus (Newcombe and Cuervo-Arango, unpublished) and age of mare and oocyte quality [9].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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2020, Journal of Equine Veterinary ScienceHorse embryo diameter is influenced by the embryonic age but not by the type of semen used to inseminate donor mares
2018, TheriogenologyCitation Excerpt :In this respect, mares bred with frozen semen are commonly inseminated post-ovulation on Day 0 (morning to afternoon) whereas fresh and cooled-transported semen are generally inseminated pre-ovulation on Day −1, with ovulation occurring at some time between the afternoon of Day −1 and the morning of Day 0. In addition to the longer ovulation-embryo flush interval in the fresh semen group, it is expected that there will be a further delay to fertilization of the oocyte following post-ovulatory insemination in the frozen semen group [8], because the sperm are thought to require 2.5–4 h to capacitate and acquire the ability to fertilize [11,12]. In contrast to previous studies, the current study included a group (G3) in which frozen semen was inseminated before and after ovulation.
Pregnancy Outcome Using Highly Concentrated, Cooled Stored Stallion Semen and Different Dilution Protocols With Autologous Seminal Plasma Before Insemination
2017, Journal of Equine Veterinary ScienceCitation Excerpt :There was a marked variability in size of embryonic vesicles because ultrasound examination was performed just once daily to detect ovulation. One mare that ovulated before insemination was excluded from the analysis of embryonic growth because embryos from postovulation inseminations are smaller in size equivalent up to about 1 day's growth [33]. In addition to three-dimensional measurements of the embryonic vesicle, more frequently performed ultrasound examinations until ovulation will provide more accurate results about the relationship between the growth of horse embryos and the SP content of the insemination dose.
Retrospective study of factors affecting multiple ovulations, embryo recovery, quality, and diameter in a commercial equine embryo transfer program
2014, TheriogenologyCitation Excerpt :This was probably due to the different moment in which AI has been performed: before ovulation with fresh semen and after ovulation with frozen semen, with a probable delay of the time of oocytes fertilization in the last case. Similar findings were reported by others that showed that postovulation inseminations produced pregnancies with smaller embryonic vesicles compared with preovulation AI, regardless of the type of semen employed [44]. In conclusion, the analysis of factors affecting ovulation rate, embryo recovery efficiency, and embryo quality performed in this study confirmed most of the results already present in the literature.
The Relationship Between the Positive Identification of the Embryo Proper in Equine Pregnancies Aged 18-28 Days and its Future Viability: A Field Study
2012, Journal of Equine Veterinary ScienceCitation Excerpt :At this time, the embryonic vesicle grows between 2.9 and 4.5 mm per day [4]. The expected mean diameter of the embryonic vesicle at 13 and 15 days post ovulation is 12.4 ± 1.8 mm and 20.1 ± 3 mm, respectively [4]. At this stage of development, a reduction in diameter equivalent to ≥2 days of growth is highly indicative of future embryo failure [3].