Elsevier

Animal Reproduction Science

Volume 164, January 2016, Pages 162-168
Animal Reproduction Science

Effect of cryopreservation technique and season on the survival of in vitro produced cattle embryos

https://doi.org/10.1016/j.anireprosci.2015.11.026Get rights and content

Abstract

Embryo cryopreservation is a major tool for conservation and propagation of genetically superior animals. However, it adversely affects the survival of embryos. The objective of this study was to determine the effects of cryopreservation technique (vitrification compared with slow freezing) and different seasons in which oocytes were obtained on the post-warming survival of in vitro produced (IVP) cattle morulae. In experiment 1, morulae (Day 6 post-IVF), obtained from abattoir-sourced oocytes during spring, summer, fall and winter over a period of 3.5 years, were subjected to either vitrification (n = 271 morulae), slow freezing (n = 281 morulae) or no freezing (control; n = 249 morulae). After warming, the morulae were cultured to the expanded blastocyst stage (Day 8 post-IVF). Data were compared using Glimmix procedure in SAS®. Blastocyst rate differed (P < 0.05) among the treatments: unfrozen control (78 ± 3.6%), vitrification (52 ± 4.6%) and slow freezing (35 ± 4.2%). The re-expansion of vitrified morulae upon warming was not correlated with subsequent blastocyst rate (r = −0.048; P > 0.05). The morulae produced during fall season had lesser (P < 0.05) cleavage and morula rates (67 ± 1.6%; Day 2 post-IVF and 22 ± 1.4%; Day 6 post-IVF, respectively) than all other seasons (74 ± 1.1 and 30 ± 1.2%, respectively). Blastocyst rate was the least (P < 0.05) when oocytes were collected during the summer season in both control and slowly frozen groups. Blastocyst development rate did not change due to season in vitrification group (P > 0.05). In conclusion, vitrification is a more desirable technique than slow freezing for cryopreservation of IVP cattle morulae. If the slow freezing method is employed, greater success can be achieved using oocytes collected in the winter and spring with a primary contributing factor being lesser morulae development if oocytes are collected in the fall and also the lesser blastocyst formation of cryopreserved morulae when oocytes are collected in the summer.

Introduction

Embryo cryopreservation is an important part of embryo transfer industry. Several hundred thousand frozen cattle embryos produced in vivo and in vitro have been exported from one country to another for purposes of subsequent transfer. Similar to other cells, conventional freezing damages mammalian embryos due to intracellular ice formation and toxic effects of permeating cryoprotectants. Moreover, embryo cryopreservation is one of the strategic approaches to conserve and improve the genetic potential of a breed. Cryobiologists aim to minimize the damage caused by intracellular ice crystal formation during the freezing and thawing cycle by using techniques that reduce this problem. The intracellular ice formation is dependent on cooling rate and surface area/volume of cells (Mazur, 1963, Mazur, 1970). Cattle embryos are conventionally frozen using a programmed slow freezing procedure. A simple and ultra-rapid freezing technique “vitrification” has been introduced (Vajta and Nagy, 2006). Both techniques are differentiated by two factors: cryoprotectant concentration and cooling rate (Leibo and Loskutoff, 1993, Massip, 2001). The slow cooling rate, in conventional freezing, increases extracellular osmolality due to ice formation that leads to an efflux of intracellular water and cell dehydration (shrinkage). Concomitantly, the solute concentration in the cytoplasm increases which decreases the intracellular freezing temperature. Cells continue to dehydrate and shrink with extracellular ice formation and the cytoplasm undergoes vitrification. In rapid cooling, the cells do not dehydrate completely, and intracellular water forms ice crystals (Mazur, 1963, Mazur, 1970). Vitrification, however, is based on the preservation of cells and tissues in a highly viscous solution of cryoprotectants to achieve a glass-like state with ultra-rapid cooling rates (>5000 °C/min) thus avoiding intracellular ice formation (Rall and Fahy, 1985, Rall, 1987, Kuwayama, 2007).

In vitrification, embryos also undergo severe dehydration (shrinkage) due to greater sucrose (non-permeating cryoprotectant) concentrations. During warming, the vitrified embryos rehydrate and regain their shape and volume. In a study with 8- to 16-cell mouse embryos, the impact of method of cryoprotectant equilibration on embryo re-expansion (volume change) and post-thaw blastocyst development was observed (Szell and Shelton, 1987). This “embryo re-expansion” is considered an indicator of embryonic viability and developmental potential (Shaw et al., 1991, Tachikawa et al., 1993, Kaidi et al., 2000, Aksu et al., 2012). A secondary objective of the current study was to validate the relationship between re-expansion of vitrified cattle morulae upon warming and subsequent blastocyst formation.

Oocyte quality and embryo survival in lactating dairy cattle are adversely affected by increased summer ambient temperatures (Gendelman et al., 2010, Braga et al., 2012, Gendelman and Roth, 2012). In vitro fertilization (IVF) has been used as an alternative to circumvent the detrimental effects of thermal stress on fertility (Rutledge, 2001, Al-Katanani et al., 2002). However, the impact of season in which the cattle embryos are produced in vitro on embryo survival with cryopreservation has not been explored extensively. Accordingly, the primary objectives of the present study were to determine the effects of common cryopreservation techniques (slow freezing compared with vitrification) and season of year (spring, summer, fall and winter) on the survival of in vitro produced cattle embryos.

Section snippets

Material and methods

The procedures used in this study were approved by the Animal Care Committee and Animal Research Ethics Board, University of Saskatchewan, Saskatoon, Canada.

Results

Blastocyst formation rates for control (not frozen), vitrification and slow freezing are depicted in Fig. 1. Blastocyst development rate was the greatest with the control (77 ± 3.6%) followed by vitrification (57 ± 4.6%) and slow freezing (35 ± 4.2%; P < 0.001) groups. Only 72.7% of morulae re-expanded upon warming and of these only 70.8% developed into blastocysts. No significant relationship was detected between re-expansion upon warming of vitrified morulae and subsequent blastocyst development rate

Discussion

In the present study, the effects of two cryopreservation techniques and four seasons on the survival and development of in vitro produced cattle morulae were investigated. This study suggested vitrification is a more desirable technique than slow freezing for cryopreservation of early developing cattle embryos. Season in which the cattle oocytes were collected, matured and fertilized in vitro influenced subsequent embryonic development. The blastocyst formation for the control and slowly

Conflicts of interest

None.

Acknowledgements

This project was funded by NSERC, EmbryoGene Network and Agriculture and Agri-food Canada. The authors would like to thank Dr. Reuben Mapletoft for his expert advice in slow freezing of the bovine embryos. We are also thankful to Dr. Kosala Rajapaksha and Dr. Dinesh Dadarwal, University of Saskatchewan, for timely help.

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