Milk-based semen diluents are known to be practical and effective in protecting equine spermatozo... more Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage. Due to complex composition of milk, the components which are beneficial or harmful to spermatozoa are unknown. To address these unknowns the effect of various milk fractions on motility of stallion spermatozoa was evaluated. The fractions tested were native phosphocaseinate (NPPC), beta-casein, whey protein concentrate (WPC), alpha-lactalbumin, beta-lactoglobulin, microfiltrate, and ultrafiltrate. The standard reference diluents were INRA 82, commercial skim milk, and Hank's salts solution supplemented with Hepes, glucose, lactose (HGLL) supplemented with BSA. After 48 and 96 h storage at 4 or 15 degrees C some milk fractions (ultrafiltrate, microfiltrate, and alpha-lactalbumin fraction) decreased spermatozoal survival. Others (beta-lactoglobulin (BL) and native phosphocaseinate) were protective. Native phosphocaseinate (NPPC) at milk concentration afforted better protection than did the standard reference diluents. The optimal concentration of beta-lactoglobulin afforted significantly better protection than did BSA. The protection afforded by native phophocaseinate was not synergistic with beta-lactoglobulin. This implies a similar mechanism of protective action of these two components. Semen diluted in HGLL supplemented with NPPC (HGLL-NPPC) or in INRA 82 and stored 24 h at 15 degrees C or 4 degrees C, respectively, produced no difference of spermatozoal motility. However, fertility of semen stored in HGLL-NPPC (60%) was higher (p < 0.05) than that stored in INRA 82 (36%).
In the horse industry, milk or milk-based extenders are used routinely for dilution and storage o... more In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen. Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed. These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.
HAL (Le Centre pour la Communication Scientifique Directe), 1997
The extent of the early stage of the Maillard-type reaction that impaired functional properties o... more The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.
HAL (Le Centre pour la Communication Scientifique Directe), Apr 21, 2009
L'instabilité du lait UHT au cours de sa conservation est une réelle préoccupation pour les i... more L'instabilité du lait UHT au cours de sa conservation est une réelle préoccupation pour les industriels et les scientifiques. Dans cette étude, les rennais de l'UMR STLO confirment la piste enzymatique, en particulier celle des protéases de Pseudomonas fluorescens qui sont capables de conserver une partie de leur activité après un traitement thermique UHT. Les chercheurs comparent la stabilité d'un lait microfiltré puis contaminé par P. fluorescens CNRZ 798 avant traitement thermique UHT et un lait témoin microfiltré et UHT exempt de toute contamination. Durant 92 jours de stockage à 20°C, les chercheurs s'intéressent aux changements physico-chimiques et biochimiques des micelles de caséines. Ils évaluent la stabilité des laits de trois manières : 1) un test au phosphate et une observation des agrégats, 2) une mesure de la taille, du potentiel zêta et de l'hydratation des micelles et 3) une analyse quantitative et qualitative de la protéolyse des laits par HPLC couplée à une analyse en spectrométrie de masse (ESI‑MS/MS). Au cours du stockage, tous les facteurs contribuant à la stabilité des micelles se détériorent dans le lait contaminé par P. fluorescens. La présence de sédiments et le résultat du test au phosphate (test de Ramsdell) permettent de constater ce phénomène. En fin de conservation, 2,4 mL de phosphate (KH2PO4 0,5 M) sont nécessaires à déstabiliser le lait témoin, alors que la déstabilisation du lait contaminé est visible avant même la réalisation du test. La granulométrie laser, le potentiel zêta et l'hydratation des micelles le confirment. Le potentiel zêta passe de ‑ 17 mV pour le témoin à ‑ 14 mV pour le lait contaminé, l'hydratation des micelles chute de 0,45 g d'eau par g de particules sèches et la taille des micelles augmente de 127 nm pour l'essai ce qui confirme leur agrégation. C'est une protéolyse accrue et complexe des caséines qui explique tous ces changements. En fin de stockage, les chercheurs mesurent dans le lait contaminé une teneur en protéines solubles (NCN) 10 fois supérieure à celle du témoin et une teneur en azote non protéique (NPN) 5 fois supérieure : + 3,4 g/kg‑1 de NCN et + 0,6 g/kg‑1 de NPN. L'aire chromatographique, huit fois supérieure dans l'essai avec P. fluorescens, illustre bien cette protéolyse. Avec la spectrométrie de masse, les auteurs montrent que le nombre de peptides du lait témoin est stable durant la conservation (environ 37) mais celui du lait contaminé passe de 51 après traitement UHT à 153 en fin de stockage. Les protéases de P. fluorescens semblent hydrolyser préférentiellement les caséines‑β puis αs1, κ et αs2. La caséine‑β est coupée en 62 liaisons situées entre les séquences 29‑69, 84‑110 et 157‑191. Sept peptides proviennent du clivage de la caséine‑κ, certains sont issus de l'hydrolyse de la liaison Phe105‑Met106. Les protéases de P. fluorescens produisent, au même titre que la chymosine, le caséinomacropeptide, expliquant ainsi la chute du potentiel zêta des micelles et leur perte d'hydratation
Milk-based semen diluents are known to be practical and effective in protecting equine spermatozo... more Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage. Due to complex composition of milk, the components which are beneficial or harmful to spermatozoa are unknown. To address these unknowns the effect of various milk fractions on motility of stallion spermatozoa was evaluated. The fractions tested were native phosphocaseinate (NPPC), beta-casein, whey protein concentrate (WPC), alpha-lactalbumin, beta-lactoglobulin, microfiltrate, and ultrafiltrate. The standard reference diluents were INRA 82, commercial skim milk, and Hank's salts solution supplemented with Hepes, glucose, lactose (HGLL) supplemented with BSA. After 48 and 96 h storage at 4 or 15 degrees C some milk fractions (ultrafiltrate, microfiltrate, and alpha-lactalbumin fraction) decreased spermatozoal survival. Others (beta-lactoglobulin (BL) and native phosphocaseinate) were protective. Native phosphocaseinate (NPPC) at milk concentration afforted better protection than did the standard reference diluents. The optimal concentration of beta-lactoglobulin afforted significantly better protection than did BSA. The protection afforded by native phophocaseinate was not synergistic with beta-lactoglobulin. This implies a similar mechanism of protective action of these two components. Semen diluted in HGLL supplemented with NPPC (HGLL-NPPC) or in INRA 82 and stored 24 h at 15 degrees C or 4 degrees C, respectively, produced no difference of spermatozoal motility. However, fertility of semen stored in HGLL-NPPC (60%) was higher (p < 0.05) than that stored in INRA 82 (36%).
In the horse industry, milk or milk-based extenders are used routinely for dilution and storage o... more In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen. Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed. These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.
HAL (Le Centre pour la Communication Scientifique Directe), 1997
The extent of the early stage of the Maillard-type reaction that impaired functional properties o... more The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.
HAL (Le Centre pour la Communication Scientifique Directe), Apr 21, 2009
L'instabilité du lait UHT au cours de sa conservation est une réelle préoccupation pour les i... more L'instabilité du lait UHT au cours de sa conservation est une réelle préoccupation pour les industriels et les scientifiques. Dans cette étude, les rennais de l'UMR STLO confirment la piste enzymatique, en particulier celle des protéases de Pseudomonas fluorescens qui sont capables de conserver une partie de leur activité après un traitement thermique UHT. Les chercheurs comparent la stabilité d'un lait microfiltré puis contaminé par P. fluorescens CNRZ 798 avant traitement thermique UHT et un lait témoin microfiltré et UHT exempt de toute contamination. Durant 92 jours de stockage à 20°C, les chercheurs s'intéressent aux changements physico-chimiques et biochimiques des micelles de caséines. Ils évaluent la stabilité des laits de trois manières : 1) un test au phosphate et une observation des agrégats, 2) une mesure de la taille, du potentiel zêta et de l'hydratation des micelles et 3) une analyse quantitative et qualitative de la protéolyse des laits par HPLC couplée à une analyse en spectrométrie de masse (ESI‑MS/MS). Au cours du stockage, tous les facteurs contribuant à la stabilité des micelles se détériorent dans le lait contaminé par P. fluorescens. La présence de sédiments et le résultat du test au phosphate (test de Ramsdell) permettent de constater ce phénomène. En fin de conservation, 2,4 mL de phosphate (KH2PO4 0,5 M) sont nécessaires à déstabiliser le lait témoin, alors que la déstabilisation du lait contaminé est visible avant même la réalisation du test. La granulométrie laser, le potentiel zêta et l'hydratation des micelles le confirment. Le potentiel zêta passe de ‑ 17 mV pour le témoin à ‑ 14 mV pour le lait contaminé, l'hydratation des micelles chute de 0,45 g d'eau par g de particules sèches et la taille des micelles augmente de 127 nm pour l'essai ce qui confirme leur agrégation. C'est une protéolyse accrue et complexe des caséines qui explique tous ces changements. En fin de stockage, les chercheurs mesurent dans le lait contaminé une teneur en protéines solubles (NCN) 10 fois supérieure à celle du témoin et une teneur en azote non protéique (NPN) 5 fois supérieure : + 3,4 g/kg‑1 de NCN et + 0,6 g/kg‑1 de NPN. L'aire chromatographique, huit fois supérieure dans l'essai avec P. fluorescens, illustre bien cette protéolyse. Avec la spectrométrie de masse, les auteurs montrent que le nombre de peptides du lait témoin est stable durant la conservation (environ 37) mais celui du lait contaminé passe de 51 après traitement UHT à 153 en fin de stockage. Les protéases de P. fluorescens semblent hydrolyser préférentiellement les caséines‑β puis αs1, κ et αs2. La caséine‑β est coupée en 62 liaisons situées entre les séquences 29‑69, 84‑110 et 157‑191. Sept peptides proviennent du clivage de la caséine‑κ, certains sont issus de l'hydrolyse de la liaison Phe105‑Met106. Les protéases de P. fluorescens produisent, au même titre que la chymosine, le caséinomacropeptide, expliquant ainsi la chute du potentiel zêta des micelles et leur perte d'hydratation
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