**1. Introduction**

The freezing process exposes the spermatozoa to thermal shock, which results in damage to the plasma membrane and acrosome [1, 2]. Various extenders have been tested in an at‐ tempt to limit cellular injury. Egg yolk is the most widely used of these extenders by artifi‐ cial insemination centres. Many demands were formulated to replace egg yolk in extenders by it's cryoprotector factor. In recent years, centrifugation techniques have enabled the isola‐ tion of the LDL(Low Density Lipoproteins) that are responsible for the cryopreservative ef‐ fect of egg yolk [3,4,5]. The incorporation of LDL in bovine extenders has given improved motility results in comparison with extenders containing egg yolk [5,6,7]. However, to ex‐ tend the use of this new LDL-based extender to insemination centres, a fertility study was essential. Fertility can either be assessed in the laboratory using *in vitro* fertilisation tests or by artificial insemination in the field [8]. The latter provides the most reliable means of as‐ sessing semen fertility following freezing and thawing. *In vitro* fertility results have already been published [6]; blastocysts were obtained after 7 days of *in vitro* culture from oocytes collected from abattoirs, matured, and then fertilised *in vitro* with spermatozoa that had been frozen-thawed in the LDL extender. An *in vitro* study is insufficient to assess the fertili‐ ty of semen that has been frozen in the LDL extender, an *in vivo* field study was therefore necessary.

*In vivo* fertility of bull semen that had been frozen-thawed in the LDL extender was as‐ sessed. A widely available, standard extender (Tris-egg yolk) was used as a control. Cows were thus inseminated with semen that had been frozen-thawed in the LDL extender; preg‐ nancy diagnoses were undertaken to assess the maintenance of fertility.

© 2013 Briand-Amirat et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1.1. Collection of semen, dilution and processing**

Two extenders were used; Tris-egg yolk extender (T-EY)): 20 ml of chicken egg yolk and LDL extender: 8% LDL (w/v) in accordance with the method described by Moussa et al. (2002) (patent n° 0100292) [5]. The extenders were thawed on the day of sampling and main‐ tained at 37°C. Three bulls belonging to an artificial insemination centre and that had been approved for public use, were used. All three had a recorded progeny. Using the Laicophos® extender, the artificial insemination centre had a non-return rate at 60-90d of 64.7% for Bull 1, 57.2% for Bull 2, and 72.2% for Bull 3. The semen was collected using an artificial vagina. To excite the bulls, they were teased with a Normandy cow for thirty minutes prior to sam‐ pling. The semen was collected into a glass tube that had been previously warmed to 37°C. Following collection, the ejaculates were immediately placed in a water bath at 37°C. Each ejaculate was divided into two equal fractions. Each fraction was immediately diluted to 100 x 106 spz/ml with the two extenders that had been previously warmed to 37°C, and then subjected to progressive cooling from 37°C to +4°C over 1h and 30 in a refrigerated unit be‐ fore being placed into straws. The semen was maintained in equilibrium for 4 hours at +4°C. The straws were held for 10 minutes at +4 cm from the surface of the liquid nitrogen (-120°C) before being immersed and then stored in liquid nitrogen (-196°C).

**2. Assesment of in vivo fertility after AI of the cows**

One hundred and ninety-three females from 83 different herds were inseminated by three inseminators with 25 years of experience. The females included in the study were from dairy or suckler herds with a Calving to First Insemination Interval (CFI) of more than 60 days, heifers over 18 months old, and first inseminations only. For each insemination, the follow‐ ing data was recorded: date of insemination, herd number, the animal's identification num‐ ber, breed, lactation or calving index, date of the previous calving if relevant, condition score, the bull used, and the extender used. The pregnancy diagnoses were conducted by re‐ cording returns to oestrus and trans-rectal palpation between the 65th and 150th day of gesta‐ tion. This data is summarised in table 3. Pregnancies can be obtained in the field following the artificial insemination of cows with semen that has been frozen and thawed in the LDL extender. However, no significant difference could be found between the LDL extender and

Fertility Results After Artificial Insemination with Bull Semen Frozen with Low Density Lipoprotein Extender

the Tris egg yolk extender in terms of the success rates of insemination (Table 4).

Motile spermatozoa (%) 58.3 ± 16.7 46.0 ± 18.2

Rapid (%) 45.3 ± 14.2 27.0 ± 12.3

Average (%) 5.7 ± 3.1 7.7± 2.5

Slow (%) 7.3 ± 3.2 11.3±4.5

Static (%) 43.7 ± 5.5 54.0± 15.1

Hyperactive (%) 5.3 ± 2.1 6.3 ± 5.9

Progressive (%) 34.7 ± 4.0 16.0 ± 6.1

VAP (µm/sec.) 83.5 ± 7.7 71.0 ± 5.3

VSL (µm/sec.) 66.6 ± 9.2 57.3 ± 6.3

LIN (%) 60.7 ± 1.5 58.3± 5.5

STR (%) 82.0 ± 1.7 79.7± 3.2

VCL (µm/sec.) 104.1± 28.6 99.9 ± 5.8

ALH (µm) 4.3 ± 0.3 4.6 ± 1.1

**Table 1.** Results of the motility of bovine spermatozoa following freezing and thawing in the LDL extender and in the Tris-egg yolk extender obtained using the Hamilton Thorne image analyser (n=3).The results given are the means ±

standard deviation of the motility characteristics recorded for the three bulls.

**LDL 8% Triladyl**

http://dx.doi.org/10.5772/51868

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### **1.2. Semen evaluation before artificial insemination in the field**

Before inseminating the cows, semen was evaluated on motility and plasma membrane in‐ tegrity. The semen was analysed using the Hamilton-Thorne sperm analyser with the CEROS 12 software program, Hamilton-Thorne biosciences, Inc, Beverly, USA. The machine had been previously configured for the analysis of bovine semen. The following parameters were studied: motility (% mobile spermatozoa), straight line velocity: VSL (μm/sec.), curvi‐ linear velocity: VCL (μm/sec.), the linearity index: LIN (= VSL/VCL x 100), amplitude of lat‐ eral head displacement: ALH (μm), and average path velocity: VAP (μm/sec). VAP, VSL, STR, and LIN provide information about the progressive movements of the spermatozoa, VCL and ALH characterise the lateral movements, and BCF (Beat Croix Frequency) pro‐ vides information about the frequency of movements.

The post-thaw percentage of motile spermatozoa was greater in the LDL extender than in the Tris-egg yolk extender (table 1). The proportion of motile spermatozoa was nearly twice as high in the LDL extender, 58.3% vs. 46% in the Tris-egg yolk (table 1). To eval‐ uate plasma membrane integrity, semen was added to an hypo-osmotic solution (100 mOsm/kg H2O). The spermatozoa was observed under a phase-contrast microscope and classified as positive or negative. Positive spermatozoa (plasma membrane intact) tail swollen and / or curled:. Negative spermatozoa (plasma membrane damaged) tail not curled. No significant difference (table 2) was found between the semen that had been frozen-thawed in the LDL and Tris-egg yolk extenders. The results of the motility analy‐ sis and plasma membrane integrity demonstrated that the semen could be used by stock breeders for artificial insemination.
