SWINE SPERM DILUTION MEDIA CONTAINING MILK PROTEIN

DESCRIPTION

This invention relates to a method for increasing and/or maintaining the viability of sperm cells in preparations for the artificial insemination of livestock. Artificial insemination (AI) has been widely accepted in the reproduction of livestock and improved reproduction in mammals. AI offers many advantages over direct mating. For example, semen collected from a single male can be used to inseminate many females, thereby reducing the number of males that are needed to maintain a population. AI also gives greater control over breeding, which results in greater reproducibility and wider and more efficient dissemination of genes or genetically determined traits. For effecting artificial insemination, semen collected from the male is first stored outside the body and then inseminated to the selected females. Therefore, the reproductive success of artificial insemination depends highly upon the satisfactory storage of semen for a reasonable long time outside the body. In general, semen for artificial insemination is stored after it has been diluted with a suitable liquid dilution medium. The kind of dilution medium and the temperature of storage have a close connection with increasing and/or maintaining the viability of the sperm cells in the dilution medium, and thus with the fertilizing capacity thereof. The processing requirements for semen used may vary according to the species. Bovine insemination requires relatively low concentrations of sperm cells, and a suitable sample may be rapidly frozen and stored for an extended period of time without adversely affecting the fertility of the sample. In contrast, swine semen cannot be processed in this way, because greater numbers of sperm cells and larger volumes of semen or diluted semen are required to inseminate females. Swine semen is generally diluted with a suitable liquid dilution medium and cooled to a storage temperature of about 17 ⁰ C. The medium serves to increase the volume of the sample to about 5 to 20 times its original volume and provides nutrients and protective substances to increase and/or maintain the viability the viability of sperm cells. Significant loss of sperm cell viability occurs after storing the semen for just a few days since the metabolism of sperm cells cannot be sufficiently inhibited. Consequently, a low conception rate is faced in artificial insemination with the use of the diluted semen stored for a period beyond five days. The relatively short time that swine sperm

cells can be stored imposes considerable constraints on the use and distribution of swine sperm cells for AI.

The present invention provides improved compositions comprising liquid dilution media for swine sperm cells. Many specific liquid media formulations are known or are available commercially for increasing and/or maintaining the viability of swine sperm cells. Typically, dilution media formulations are provided in solid form (powder), and are dissolved in water for use. Standard formulations can be found in the art. For example, J.Gadea reviewed the latest knowledge on swine semen dilution media used in artificial insemination procedures. The requirements of an effective fresh semen dilution medium were discussed and currently available dilution media were compared (see http://www.engormix.com/semen_extenders_used_in_e_articles_8 7_POR.htm). A swine sperm cell dilution medium is generally one containing salts, an energy source (e.g. glucose), one or more antibiotics and at least one buffer (e.g. 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES)) so that the resulting solution has a pH value between 6 and 7.5. Additional components may include ethylene diamine tetraacetic acid (EDTA) and bovine serum albumin (BSA). The resulting mixture of substances, when dissolved in water, should result in a solution with an osmotic pressure of between 220 to 380 milliosmoles. Bovine serum albumin (BSA) is a protein that is widely used in compositions for increasing and/or maintaining the viability of sperm cells. BSA stabilizes macromolecules in solution of water and is also widely used in cell culture media. BSA also binds many toxic substances and stabilizes the pH of the dilution medium. The properties of BSA make it a particularly suitable protective substance for increasing and/or maintaining the viability of sperm cells. However, the use of BSA has two major drawbacks. Firstly, BSA is isolated and purified from bovine blood serum. Therefore, it poses a potential threat since the use of certain bovine products, most notably blood products, are related to the occurrence of Bovine Spongiphoπn Encephalopathy (BSE). Secondly, the isolation and purification of BSA is a costly process, and does not always result in a product with reliable quality and performance. Consequently, the cost price of BS A is relatively high and makes up a major part of the cost of dilution media for

swine sperm cells. So far there is no good alternative to BSA for applications in swine sperm cell dilution media. To provide the AI industry with a more economical and more appropriate replacement of BSA is a major object of the present invention.

The present invention relates to the application of protein from cow's milk - primarily as replacement for BSA - in liquid swine sperm cell dilution media. Milk protein is a relatively cheap and widely available source of protein. Milk protein is the name for a collection of proteins that can be isolated from milk, or from whey, a by-product of cheese manufactured from cow's milk. It is typically a mixture of immuno-globulins, β-lactoglobulin, α-lactalbumin, serum albumin, and many other proteins, which are soluble in their native form. The use of milk proteins in dilution media for sperm was already described in 1953 by M.R.E.J. Cassou (patent FR 1043684). Cassou described the use of fat free milk to dilute cow sperm. Of a more recent date are the extensive studies of Batellier et al. (Theriogenology 48 (1997) pp 391-410; patent EP 0969850B1). These studies deal primarily with the use of casein fractions of milk in dilution media for horse sperm. Of more recent date is the work of L. de Backer en S. Poncelet (patent NL 1024129A). In this patent the use of fat free milk powder (of brand Gloria produced by Nestle) in dilution media for horse sperm is described. Finally, the study of M.T. Pellicer-Rubio et al. describes the use of milk containing dilution media for goat sperm (M.T. Pellicer-Rubio, T. Magallon and Y. Combarnous. Biology of Reproduction 57 (1997) 1023-1031). In contrast to the above- mentioned examples (skim) milk or casein (native phosphocasein) has not successfully been applied for preserving swine sperm cells at 17 ⁰ C. Use of (skim) milk or casein has been reported though for cryopreservation of swine sperm cells. But this has not widely been applied successfully in the art.

Various components of milk are known to be both beneficial and detrimental to sperm viability. Research done in recent years showed that caseins, the major proteins of milk, appear to be responsible for the protective effect of milk on sperm in preservation media In a similar manner as egg yolk protein, caseins decreased the binding of members of a family of lipid-binding proteins (so-called BSP proteins that induce cholesterol and phospholipid removal from the sperm

97 membrane) to sperm and sperm lipid loss, while maintaining sperm motility and

98 viability during storage. Caseins prevent the detrimental effect of BSP proteins on

99 the sperm membrane during sperm preservation (A.Bergeron , Y.Brindle , P.

100 Blondin , P.Manjunath, Biology of Reproduction 77, (2007) ppl20-126; A.

101 Bergeron, P. Manjunath. Molecular Reproduction and Development. Volume 73,

102 2007, pp 1338 - 1344). Surprisingly, Batellier et al. found that whey protein

103 concentrate, milk micro filtrate and ultra filtrate decreased stallion sperm

104 survival after 48 and 96 h storage at 4 or 15 ⁰ C (F. Batellier , M. Magistrini , J.

105 Fauquant , E. Palmer. Theriogenology 48 (1997) pp. 391-410). The use of milk

106 serum protein or whey protein would thus be unlikely to be useful for those in the

107 art.

108 Notwithstanding the successful application of milk containing or casein

109 containing dilution media at temperatures at or around 5 ⁰ C for sperm cells of

110 several species, such as stallion and bull, a similarly successful application for

111 liquid dilution media for swine sperm cells has never been reported. Swine sperm

112 cells are very different from many other species' sperm cells in that they are

113 extremely vulnerable to cold shock. For this reason on may not assume that a

114 sperm dilution medium that is applicable for a certain kind of mammal will also

115 be applicable for all mammals so that a one skilled in the art may easily translate a

116 successful dilution media from one species to another. Swine sperm cells have a

117 different composition of the phospholipids in the membrane when compared to

118 many other mammals. A low cholesterol/phospholipid ratio and an asymmetrical

119 distribution of cholesterol within the membrane render swine sperm cells very

120 susceptible to cold temperatures resulting in increased membrane permeability

121 and loss of controlled membrane processes. Hence, rapid cooling of ejaculates to

122 15 ⁰ C or cooling below 15 ⁰ C results in loss of viability. To avoid this cold shock,

123 pre-diluted ejaculates are better left at temperatures above 15 ⁰ C for several hours

124 to induce cold resistance. In practice, semen is collected in isolated cans to avoid

125 contact with colder surfaces and subsequent dilution is done with a dilution

126 medium at 35-37 ⁰ C after which the semen is allowed to cool down gradually to

127 17 ⁰ C. Further storage of diluted semen is done at 17 ⁰ C, at which temperature

128 semen metabolism is reduced, a condition necessary to extend storage time.

129 Storage below 1 2°C has a negative effect on motility and membrane integrity in

130 vitro (P. Vyt. Thesis. Examination and storage of liquid porcine semen. Faculty of

131 Veterinary Medicine, Ghent University 2007).

132 The present invention relates to the application of protein from cow's milk -

133 primarily aimed as replacement for BSA - in liquid swine sperm cell dilution

134 media, in particular protein derived from whey. Application of milk protein

135 (neither casein, nor other protein from milk or whey), to replace BSA in

136 applications where BSA exerts its role as mentioned above and where it replaces

137 the function of BSA, has not been reported for swine sperm cell dilution media.

138 Surprisingly, we found a particular fraction of proteins derived from whey that

139 showed positive properties with respect to increasing and/or maintaining the

140 viability of swine sperm cells in a liquid dilution medium. This particular fraction

141 of proteins derived from whey was named Porex, for sake of short description.

142 Porex showed a superior performance over that of BSA with respect to increasing

143 and/or maintaining the viability of swine sperm cells as shown by motility during

144 sperm cell storage and as shown by farrowing rate and, more importantly, litter

145 size.

146 Porex is produced from whey concentrate. Pertaining to the current invention is

147 production of Porex by membrane filtration under moderate conditions. There are

148 several methods in the art to isolate and purify water-soluble milk or whey

149 proteins. Of these, filtration is a well-know and widely applied technique

150 (Rombaut et al. J. Dairy Sci. 90 (2007) pp 1662-1673). Those skilled in the art of

151 fluid dairy filtration may easily apply existing filtration techniques. To produce

152 Porex, whey concentrate is first micro filtrated using a membrane filter with pore

153 size of 200 micrometer. This allows all smaller bioactive protein to pass, while

154 capturing any particulate from microbes or denatured protein. The resulting

155 bioactive protein is further purified by ultra filtration. Ultra filtration is

156 characterized as having a molecular weight cut-off range (MWCO) from about

157 3000 to 100,000 Dalton. Porex is produced using a MWCO of 10,000 Dalton. A

158 filter with pore size of 10,000 Dalton allows minerals and lactose from the whey

159 to pass while the bioactive protein is further concentrated. The concentrated

160 protein is then spray dried. An absolute prerequisite to preparing Porex

161 appropriate for the current invention is maintaining pH and moderate temperature

162 during processing of the whey. Prior to micro filtration the whey concentrate is

163 held at 66 ⁰ C for 3 minutes. Thereafter the temperature is lowered and kept at 22

164 ⁰ C. Porex purified in this way is made up of mainly β-lactoglobulin, α-

165 lactalbumin and glycomacropeptide. In addition, Porex contains also the less

166 abundant proteins that occur in much lower concentration in milk, such as

167 lactoferrin en lactoperoxidase. These proteins play an important role in the

168 proposed liquid swine sperm cell dilution media. A further important

169 characteristic of Porex is absence of free or unbound fat and minerals. Porex only

170 contains minimal amounts of fat and minerals bound to protein.

171 The above-mentioned process to produce Porex leads to a highly purified

172 unmodified and non-denatured, native protein product which is soluble in water,

173 up to 30 gram per 100 milliliter. A typical analysis shows that dry Porex contains

174 97% protein, 2.3% ash, and 0.3% fat. The protein fraction contains 46 ± 3% β-

175 lactoglobulin, 16 ± 2% α-lactalbumin, and 26 ± 2% glycomacropeptide. The

176 10% remaining fraction contains, among the hundreds of other proteins, bovine

177 serum albumin, lactoferrin, and lactoperoxidase.

178 The present invention provides improved compositions comprising dilution media

179 for swine sperm cells containing Porex that functions in protecting the sperm cell

180 by the concerted action of several proteins. One of these proteins is β-

181 lactoglobulin which binds a wide range of substances among which many metals

182 and hydrophobic molecules such as long-chain fatty acids, retinoids, steroids, and

183 cholesterol. The binding properties of β-lactoglobulin may aid in removing

184 oxidized lipids, that otherwise would keep the chain of oxidation running and

185 would damage the sperm cell membrane. Another protein present in Porex is α-

186 lactalbumin. This protein has antibacterial and anti-oxidative properties and binds

187 metals. Glycomacropeptide has antiviral and antibacterial properties. Bovine

188 serum albumin can bind reversibly a variety of substances. It can also sequester

189 free radicals and inactivate various toxic lipophilic metabolites. Albumin has a

190 high affinity for fatty acids, hematin, bilirubin and a broad affinity for small

191 negatively charged aromatic compounds. It forms covalent adducts with pyridoxyl

192 phosphate, cystein, glutathione, and various metals. Lactoferrin is an iron binding

193 protein that exerts multiple effects, including acting as an antimicrobial agent.

194 Lactoperoxidase has been identified as an antimicrobial agent. It has proven to be

195 both bactericidal and bacteriostatic to a wide variety of micro-organisms. The

196 concerted action of these proteins may be aided by many other proteins that are

197 present in milk serum.

198 The present invention provides improved compositions comprising dilution media

199 for swine sperm cells containing Porex that functions in protecting the sperm cell

200 among others against many toxic substances and against oxidation. There are

201 several toxic substances that may damage the sperm cell. These are metabolic

202 products from dead sperm cells and other cells such as bacteria and leucocytes,

203 toxic products produced by bacteria, trace heavy metals, and free radicals such as

204 reactive oxygen species and reactive nitrogen species.

205 During spermatogenesis, after ejaculation and suspension in dilution media, and

206 after introduction into the female genital tract, the sperm cell can be damaged by

207 free radicals (reactive oxygen species and reactive nitrogen species). In the

208 ejaculate, in the diluted medium and in the female tract the sperm cells face -

209 coming from the male reproductive organ - new biochemical environments where

210 different oxidative regimes are present. Porex serves here to protect sperm cells

211 from oxidation. Most important, Porex protects oxidation of cell membrane

212 components by the anti-oxidative action of several proteins among which β-

213 lactoglobulin and lactoperoxidase.

214 Bacteria and bacterial toxins originate mostly from the prepuce, from semen

215 manipulation or from the water used in the extender preparation. Also, the

216 chemicals used to prepare dilution media are not free from bacteria and bacterial

217 contaminants. Depending on the species, bacteria have deleterious effects on

218 semen quality, namely depressed motility, cell death and agglutination, either by

219 direct effect on the spermatozoa or by acidifying the environment. Swine sperm

220 cells are extremely vulnerable to bacterial toxic products. Swine sperm cells have

221 a different composition of the phospholipids in the membrane when compared to

222 many other mammals which make them also more susceptible to bacterial toxins.

223 Hydrophilic toxins may enter the cell by passing the membrane more easy. Most

224 noteworthy, due to this unusual characteristic of swine sperm cells (being so

225 sensitive to bacterial toxins) a swine sperm motility inhibition assay was

226 developed into an assay useful for specific detection of mitochondria damaging

227 toxins (D. Hoornstra, M. A. Andersson, R. Mikkola, M. S. Salkinoja-Salonen.

228 Toxicology in Vitro, 17 (2003), pp 745-751 ). BS A is believed to have a positive

229 influence on sperm survival among others due to the absorption of metabolic

230 bacterial products from the dilution medium, hi the same way, but likely more

231 effective due to the presence of different proteins, Porex protects swine sperm

232 cells from the action of bacterial toxins.

233 The present invention provides a composition comprising a sperm cell medium

234 particularly useful for swine sperm cells. Enhanced viability of stored sperm is

235 shown by motility determinations and - more decisively for the one skilled in the

236 art — by assessment of farrowing rates and litter size following large-scale AI in an

237 industrial setting.

238 For the purpose of exemplification, the composition of the present invention will

239 be hereinafter illustrated by their advantageous use in the field of preserving

240 swine sperm cells for insemination. Following is a description by way of example

241 of methods of carrying the invention into effect. Those affiliated with artificial

242 insemination of swine females know the art of preparing media, determination of

243 general and progressive motility using computer assisted analysis, dilution and

244 storage of semen, and insemination of swine females. Further necessary

245 information is given with the three examples below.

246 Concentrations of components in liquid medium are expressed in terms of the

247 number of units mass per liter. One of skill in the art would appreciate that the

248 medium of the present invention may be prepared in any volume, and the

249 invention is not intended to be limited to media prepared in one liter volumes.

250 Further, the present invention is not intended to be limited to the formulation

251 given. Other formulations containing any amount of Porex or any protein isolated

252 from milk or from Porex, but not casein, may be applied as well.

253 Example 1. Swine semen dilution medium was prepared by adding 980 milliliter

254 of water to: a combination of 27,4 gram glucose, 8 gram sodium citrate, 2,4 gram

255 EDTA, 1,2 gram sodium bicarbonate, 0,4 gram potassium chloride, 0,6 gram

256 gentamycin, 9 gram HEPES, and 1 gram Porex. This composition is named Porex

257 dilution medium. As reference - to compare with a commercially available

258 dilution medium - was used the widely accepted dilution medium Beltsville

259 Thawing Solution (BTS). BTS medium was prepared by adding 980 ml water to

260 a combination of 36,4 gr glucose, 6 gram sodium citrate, 1,25 EDTA, 1,25 sodium

261 bicarbonate, 0,75 gram potassium chloride and 0,6 gram gentamycin. Semen was

262 collected from 12 randomly selected, sexually mature boars. Sperm was diluted

263 with Porex dilution medium and BTS, and general motility and progressive

264 motility was assessed at day 1, 3, 5, 9 after dilution of the semen.

265 The general motility data are as follows. For BTS the %-age of sperm cells with

266 good general motility found were 71.4, 70.4, 68.9, 47.9 for day 1, 3, 5, 9 after

267 dilution, respectively. For the Porex dilution medium the %-age of sperm cells

268 with good motility were 85.0, 82.9, 80.5, and 69.6 for day 1 ,3, 5, 9 after dilution,

269 respectively.

270 The progressive motility data are as follows. For BTS the %-age of sperm cells

271 with good progressive motility were 42.3, 37.8, 36.2, 24.3 for day 1, 3, 5, 9 after

272 dilution, respectively. For the Porex dilution medium the %-age of sperm cells

273 with good progressive motility were 76.4, 73.4, 68.5, and 57.5 for day 1, 3, 5, 9

274 after dilution, respectively.

275 Example 2. This example refers to semen collection, dilution and distribution

276 carried out by a commercially AI station, and AI at 8 different swine farms. Swine

277 semen dilution medium was prepared by adding 980 milliliter of water to a

278 combination of 27,4 gram glucose, 8 gram sodium citrate, 2,4 gram EDTA, 1,2

279 gram sodium bi carbonate, 0,4 gram potassium chloride, 0,6 gram gentamycin, 9

280 gram HEPES, and 1 gram milk Porex. During a period of 4 weeks (May 2006)

281 semen was collected from 28 sexually mature boars from 5 different swine races

282 (Pietrain, York, Dalpo, Primeur, Hampshire). The sperm was diluted with the

283 Porex sperm dilution medium and 890 females were inseminated. The average

284 farrowing rate found was 92.0% and the average litter size found was 13.8. 285

286 Example 3. This example refers to semen collection, dilution and distribution

287 carried out by a commercially AI station and AI at 8 different swine farms. Swine

288 semen dilution medium was prepared by adding 980 milliliter of water to a

289 combination of 27,4 gram glucose, 8 gram sodium citrate, 2,4 gram EDTA, 1,2

290 gram sodium bi carbonate, 0,4 gram potassium chloride, 0,6 gram gentamycin, 9

291 gram HEPES, and 1 gram Porex. During a period of 4 weeks (June 2006) semen

292 was collected from 28 sexually mature boars from 5 different swine races

293 (Pietrain, York, Dalpo, Primeur, Hampshire). The sperm was diluted with the

294 Porex dilution medium and 930 females were inseminated. The average farrowing

295 rate found was 91.0% and the average litter size found was 13.5. 296

297 Examples 2 and 3 give more decisive information than example 1 for those in the

298 art since farrowing rate and litter size are the economic parameters in the swine

299 business. Since farrowing rate and litter size vary depending on farm

300 management, a comparison of dilution media on the same farms was done. The

301 litter size found was 0.6 higher than the litter size found with standard BTS

302 dilution medium as described in example 1. The average farrowing rate found was

303 4% higher with Porex dilution medium compared to BTS dilution medium.

304 After the work described in the examples mentioned above, 80.000 inseminations

305 were done using the Porex dilution medium described in examples 2 and 3 during

306 the period of July 1, 2006 till December 25, 2007. An average farrowing rate of

307 94.0 % and an average litter size of 14.1 was found. The number of pigs born

308 alive was on average 13.4. The number of pigs born alive is the most important

309 economic indicator in the pig growing business. The number of pigs born alive in

310 The Netherlands using standard dilution medium (BTS) can be taken from the

311 data published by Agrovision based on the software Pigmanager

312 (http://www.agrovision.nl/files/ksp20062007website01.pdf). For the period July

313 1, 2006 - June 302007 a farrowing rate of 86%, litter size of 13.1 and number of

314 pigs born alive of 12.5 was found. This shows that the Porex dilution medium is a

315 more economic dilution medium than the widely used BTS.

316 In accordance with the embodiment of the present invention, we have found that

317 the viability of swine sperm cells can be increased and/or maintained by dilution

318 in media that contain Porex so that the fertilizing capacity of the sperm cells can

319 be increased and/or maintained for an extended period of time. Thus we have

320 found more economical and superior dilution media by replacing BSA with milk

321 serum protein with brand name Porex.

322

323 Conclusions

324 Although only an exemplary embodiment of the invention has been described in

325 detail above, those skilled in the art will readily appreciate that many

326 modifications are possible without materially departing from the novel teachings

327 and advantages of this invention. Accordingly, all such modifications are intended

328 to be included within the scope of this invention as defined in the following

329 claims.

330