Elsevier

Theriogenology

Volume 65, Issue 6, 1 April 2006, Pages 1007-1015
Theriogenology

In vitro production of bovine embryos using sex-sorted sperm

https://doi.org/10.1016/j.theriogenology.2005.07.007Get rights and content

Abstract

The objective of this study was to investigate the suitability of sex-sorted sperm for producing viable in vitro embryos for subsequent transfer into recipient cows and heifers on commercial dairy farms. From August 2002 to June 2003, ovaries were collected from 104 producer-nominated Holstein donor cows on seven Wisconsin farms via colpotomy or at slaughter. Oocytes (N = 3526) were aspirated from these ovaries, fertilized 22 ± 0.2 h later, and cultured to the morula or blastocyst stage. The fluorescence-activated cell sorting (“Beltsville”) approach was used to produce (primarily) X-bearing sperm from the ejaculates of three young Holstein sires, and 365 transferable embryos were produced. On average, 3.6 ± 0.3 (means ± S.E.M.) transferable embryos were produced per donor, including 1.4 ± 0.2 (Grade 1), 1.5 ± 0.2 (Grade 2), and 0.7 ± 0.1 (Grade 3) embryos. Number of usable oocytes per donor (33.9 ± 3.3) and percent cleavage (51.1 ± 1.9) were significant predictors of the number of blastocysts that developed. Mean conception rates for the resulting in vitro embryos were 34.2 ± 1.6% in yearling heifer recipients and 18.2 ± 0.7% in lactating cow recipients. Additional oocytes (N = 3312) from ovaries of anonymous donors (N unknown) collected at a commercial abattoir were fertilized using unsorted sperm, and the percentage of these that developed to blastocyst stage (20.1 ± 2.9) was greater (P < 0.05) than the corresponding percentage (12.2 ± 2.3) achieved with sex-sorted sperm using oocytes (N = 1577) from the same source. In summary, we inferred that in vitro embryo production may be a promising application of sex-sorted sperm in dairy cattle breeding, but that the biological causes of impaired embryo development in vitro and compromised conception rates of transferred embryos should be further investigated.

Introduction

Technologies for sorting bovine sperm into X- and Y-bearing fractions could have a major impact on breeding programs in dairy cattle and other livestock species; several recent papers [1], [2], [3], [4], [5], [6], [7] have addressed possibilities, limitations, and potential applications. If cost-effective, procedures that isolate X-bearing sperm could have important economic consequences through enhanced availability and affordability of replacement dairy heifers. Hohenboken [2] hypothesized that the average genetic merit of replacement heifers could be improved through the use of sex-sorted semen. Likewise, Van Vleck et al. [8], [9] calculated that producers could benefit financially from sex-sorted sperm, even if it cost twice as much as unsorted sperm. Although sex-sorting of sperm has been the focus of many research projects using a variety of approaches [10], [11], [12], this technology has not yet become cost-effective for routine use on commercial farms [1], [13]. Presently, the only proven method for sex-sorting of sperm is the fluorescence-activated cell sorting approach of Johnson et al. [14], [15], [16], [17]. The so-called “Beltsville Sperm Sexing Technology” is based on a 3.8% difference in DNA content of X- versus Y-bearing bovine sperm. When the DNA of sperm is stained with a fluorescent dye and subjected to flow cytometry, a brighter fluorescence is emitted from the edge of the sperm head compared with the more transparent flat side. Proper sperm orientation can be controlled during flow-cytometric sorting using a beveled nozzle. A charge is applied to each droplet, the droplets are deflected as they pass between two charged plates, and droplets with no charge become waste.

Like sperm-sorting, technology for in vitro production (IVP) of bovine embryos has also encountered many challenges on the path toward widespread commercial application. However, IVP technology may be more useful when combined with sperm-sorting technology [18]. Amann [1] discussed the potential for “tailoring” sperm sorting to the needs and limitations of IVP. Although “niche” applications of these technologies to valuable breeding stock may benefit a few selected individuals, widespread applications on commercial farms will require development of cost-effective breeding programs that can be implemented within the routine management of a dairy or livestock operation. In the case of dairy operations, “low-cost” programs that utilize known (e.g., high genetic merit) or anonymous cull cows as donors may be cost-effective, provided that the timing of the embryo production schemes can be successfully matched with that of the controlled breeding programs on participating farms [19].

The primary objective of this study was to examine the suitability of sex-sorted sperm in IVP of dairy embryos for subsequent transfer into recipient cows or heifers on commercial farms. More specifically, we sought to determine the number of oocytes that could be recovered, the rate of blastocyst development, and the number (and quality) of embryos that could be produced in a routine in vitro embryo production system utilizing sex-sorted bovine sperm. When possible, we compared these variables with corresponding measurements from unsorted (control) sperm.

Section snippets

Materials and methods

Between August 2002 and June 2003, ovaries were recovered from Holstein donors (N = 104) on seven commercial dairy farms in Wisconsin. All farms were within 4 h driving time from the IVP laboratory (BOMED, Inc., Madison, WI, USA). Donor animals were selected by these producers from the pool of animals to be culled (in any given month) based on superior genetic merit and/or phenotypic performance. Virtually all donors were “involuntary culls”, i.e., productive, profitable cows whose removal from

Results

Twenty-four laboratory replicates were completed with unsorted sperm; due to limited sperm availability, only 19 replicates were carried out using sex-sorted sperm. As shown in Table 1, the blastocyst development rate from oocytes of anonymous donors fertilized with sex-sorted or unsorted sperm differed (P < 0.05), with significantly more blastocysts produced from the unsorted sperm.

Table 2 shows a detailed summary of data from producer-nominated cows and heifers used as donors in the present

Discussion

The use of flow-cytometrically sorted sperm in IVP systems has been investigated in several previous studies [30], [31], [32], and results of the present study generally confirmed those findings. Viable embryos can be cultured to a transferable stage (day 7 or 8) successfully, but a greater number of usable oocytes are needed to develop the same number of transferable embryos when sex-sorted sperm are used.

Given that cleavage rates were similar for IVP with sex-sorted or unsorted sperm, it

Acknowledgements

Financial support was generously provided by the University of Wisconsin Office for Corporate Relations and the Wisconsin Department of Agriculture, Trade, and Consumer Protection. Semen was provided by Accelerated Genetics, Inc., and XY, Inc., and BOMED, Inc. provided in vitro embryo production services. Technical assistance was provided by Val Schutzkus, Lee Matthews, Rick Monson, and Milo Wiltbank. Special thanks to the seven dairy farms (Crave, Holterman, Keller, Larson, Rickert, Ruedinger,

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