Assessment of industrial lipases for flavour development in commercial Idiazabal (ewe's raw milk) cheese

Three commercial lipases (lipases A1 and A2 were pregastric lipases and lipase A3 was a fungal lipase) from three different manufacturers were studied to develop the characteristic pungent flavour of Idiazabal (sheep's raw milk) cheese in experimental productions (50 L vats), both in summer and in winter. In the experimental productions, all lipases significantly increased the concentration of total free fatty acids (FFA), both after 90 and 180 days of ripening. Lipases A1 and A2 increased primarily the concentration of short-chain FFA (C4, C6 and C10), whereas C16:0 and C18:1 were the main FFA obtained with lipase A3. Cheeses made with no lipase had the lowest concentrations of total FFA, with C16:0 and C18:1 as the major FFA. The percent of short-chain FFA was 70–80% of the total for lipases A1 and A2, but 30–40% in cheeses made with lipase A3 or with no lipase. Sensory analysis was performed after 90 and 180 days of ripening. Cheeses made with lipases A1 or A2 or with lamb rennet paste (which contains pregastric lipase and was used as control for sensory analysis) had significantly higher scores in 6 of the 22 attributes analyzed (odour and flavour intensity, sharp odour, pungent flavour and rennet odour and flavour) than cheeses made with lipase A3 or with no lipase added. Lipase A1 was selected to conduct commercial cheese productions (200 L vats) made by artisan cheese makers adding 94 lipase units (LU)/50 L (high amount) or 8 LU/50 L (low amount). Control cheeses were made with lamb rennet paste having comparable amounts of lipolytic activity. After 90 and 180 days of ripening, cheeses made with the same amount of lipase (either as lipase A1 or present in lamb rennet paste) were indistinguishable from each other, both from the sensory and analytical points of view (comparable amounts of total FFA, and percent FFA composition). Those with low amount of lipase were rated as ‘mild’, whereas those with high amount of lipase were rated as ‘strong’. An industrial production (5000 L vat) with 100 LU/50 L yielded cheeses of ‘intermediate strength’ and intermediate levels of lipolysis. A linear correlation was observed between percent of short-chain FFA and the score for pungent flavour, for all amounts of lipase used in this study. We conclude that lipase A1 is an adequate commercial lipase to develop the characteristic flavour of Idiazabal cheese, both in artisan and industrial fabrications.     

Evaluation of amino acid-decarboxylative microbiota throughout the ripening of an Italian PDO cheese produced using different manufacturing practices

Aim:  To investigate the presence of biogenic amines (BAs) in Montasio cheese produced by using different cheese manufacturing practices.
Methods and Results:  Three batches of Montasio cheese were made in the following way: batch A using raw milk and natural milk culture, batch B with thermized milk and natural milk culture and batch C with thermized milk and natural milk culture added of a commercial starter culture. During 120 days of ripening analyses were performed for microbial counts and BA content; indeed, the potential to produce BAs was screened in lactic acid bacteria and Enterobacteriaceae isolates. At the end of ripening, the total BA contents of cheeses from batches A, B and C were 166·3, 207·3 and 29·8 mg kg⁻¹, respectively. Amino acid decarboxylase activity was widespread among isolates.
Conclusions:  The BA content of Montasio cheese from the three batches was below the threshold proposed as potentially toxic. The highest BA content was found in cheese produced using thermized milk and natural milk culture; therefore, the thermal treatment of milk was not enough by itself to reduce the counts of decarboxylase-positive bacteria in cheese. The use of selected starters guaranteed a low BA content in Montasio cheese.
Significance and Impact of the Study:  The study of the effects of some technological processes on the incidence of decarboxylative microbiota in 'protected denomination of origin' cheeses could provide useful information on the hygienic risk related to their production.     

Optimized enrichment for the detection of Escherichia coli O26 in French raw milk cheeses

Aims: Our main objective was to optimize the enrichment of Escherichia coli O26 in raw milk cheeses for their subsequent detection with a new automated immunological method. Methods and Results: Ten enrichment broths were tested for the detection of E. coli O26. Two categories of experimentally inoculated raw milk cheeses, semi‐hard uncooked cheese and ‘Camembert’ type cheese, were initially used to investigate the relative efficacy of the different enrichments. The enrichments that were considered optimal for the growth of E. coli O26 in these cheeses were then challenged with other types of raw milk cheeses. Buffered peptone water supplemented with cefixim–tellurite and acriflavin was shown to optimize the growth of E. coli O26 artificially inoculated in the cheeses tested. Despite the low inoculum level (1–10 CFU per 25 g) in the cheeses, E. coli O26 counts reached at least 5·10⁴ CFU ml^?1 after 24‐h incubation at 41·5°C in this medium. Conclusions: All the experimentally inoculated cheeses were found positive by the immunological method in the enrichment broth selected. Significance and Impact of the Study: Optimized E. coli O26 enrichment and rapid detection constitute the first steps of a complete procedure that could be used in routine to detect E. coli O26 in raw milk cheeses.     

Predictive formulae for goat cheese yield based on milk composition

Prediction of the yield and quality of different types of cheeses that could be produced from a given type and/or amount of goat milk is of great economic benefit to goat milk producers and goat cheese manufacturers. Bulk tank goat milk was used for manufacturing hard, semi-hard and soft cheeses (N = 25, 25 and 24, respectively) to develop predictive formulae of cheese yield based on milk composition. Fat, total solids, total protein and casein contents in milk and moisture-adjusted cheese yield were determined to establish relationships between milk composition and cheese yield. Soft, semi-hard and hard cheeses in this study had moisture contents of 66, 46 and 38%, respectively, which could be used as reference standards. In soft cheese, individual components of goat milk or a combination of two or three components predicted cheese yield with a reasonably high correlation coefficient (R² = 0.73–0.81). However, correlation coefficients of predictions were lower for both semi-hard and hard cheeses. Overall, total solids of goat milk was the strongest indicator of yield in all three types of cheeses, followed by fat and total protein, while casein was not a good predictor for both semi-hard and hard cheeses. When compared with moisture-adjusted cheese yield, there was no difference (P > 0.05) in predicting yield of semi-hard and hard goat milk cheeses between the developed yield formulae in this study and a standard formula (the Van Slyke formula) commonly used for cow cheese. Future research will include further validation of the yield predictive formulae for hard and semi-hard cheeses of goat milk using larger data sets over several lactations, because of variation in relationships between milk components due to breed, stage of lactation, season, feeding regime, somatic cell count and differences in casein variants.     

Association of Yersinia enterocolitica with the manufacture of cheese and occurrence in pasteurized milk.

Raw milk in southern Ontario frequently contains Yersinia enterocolitica. The potential for transmission of this organism by cheese manufactured from unpasteurized milk was evaluated by examination of milk and cheese curd samples from cheese manufacturing plants and finished cheddar and Italian cheeses. The incidence of Y. enterocolitica was lower in cheese curd samples (9.2%) than in raw milk (18.2%). Most of the curd samples showed a positive phosphatase test, indicating production from raw milk. One curd sample yielded Y. enterocolitica after 4 weeks of storage at 4 degrees C but was negative after 8 weeks. All samples of cheddar and Italian cheeses, most of which showed a positive phosphatase test, were negative for Y. enterocolitica. One out of 265 samples (0.4%) of pasteurized fluid dairy products contained Y. enterocolitica.     

Winter feeding systems and dairy cow breed have an impact on milk composition and flavour of two Protected Designation of Origin French cheeses

This study investigates the effects of two feeding systems and two dairy cow breeds on milk yield and composition, physical and sensorial properties of Camembert and Pont-l’Evêque cheeses. The experiment consisted of a 2 × 2 factorial arrangement of treatments. A low energy grass diet with only 15% of concentrate (LowGS) was compared with a high-energy maize silage diet with 30% concentrate (HighMS). Thirty-four Holstein (Ho) and 34 Normande (No) cows in early lactation were assigned to one of two feeding systems for a 6-week period. Cows on the LowGS feeding system had lower milk yield, fat and protein content. In both feeding systems, No cows had lower milk yields but higher milk protein contents than Ho cows. The LowGS feeding system altered milk fatty acid (FA) composition by reducing saturated FA. Breed had only a small impact on milk FA. Concerning milk coagulating properties, only the firmness was reduced by the LowGS feeding and the Ho breed. The effects of breed and feeding system on the protein content of cheeses were more marked in Camembert cheese than in Pont-l’Evêque cheese. However, the Camembert cheese from Ho-LowGS was, in fact, characterized especially by lower protein content. LowGS feeding system and No breed produced more yellow cheeses. Feeding systems had limited effects on the firmness of Camembert and Pont-l’Evêque cheeses measured by penetrometry. In sensory analysis, Ho breed and LowGS feeding produced a Camembert cheese with a more melting texture in the mouth due to the increase of spreadability index and of proteolysis. The type of cheese also had an influence: the effects were more important on Camembert cheese than on Pont-l’Evêque cheese. Only the Ho-LowGS treatment produced a very specific Camembert cheese different from the others. The feeding systems and breed of dairy cow have no determinant effect on PDO (protected designation of origin) Camembert and Pont-l’Evêque cheeses, especially regarding taste. In this kind of trial, despite the effects of feeding systems and breed on milk composition, the role of cheese ripening and microbiology appears to be of considerable importance.     

Influence of incubation temperature and time on resistant starch type III formation from autoclaved and acid-hydrolysed cassava starch

Raw cassava starch, having 74.94 and 0.44 g/100 g resistant starch type II and III (RS II and RS III), respectively, was autoclaved at 121 °C in water, 1, 10 or 100 mmol/L lactic acid. The formation of RS III was evaluated in relation to variable incubation temperature (−20 to 100 °C), incubation time (6–48 h) and autoclaving time (15–90 min). Negligible to low quantities of RS III (0.59–2.42 g/100 g) were formed from autoclaved starch suspended in 100 mmol/L lactic acid, whereas intermediate to high quantities (2.68–9.97 g/100 g) were formed from autoclaved starch suspended in water, 1 or 10 mmol/L lactic acid, except for treatments with water or 10 mmol/L lactic acid incubated at 100 °C for 6 h (1.74 g/100 g). Autoclaving times corresponding to maximum RS III contents were 15 and 45 min for water and 10 mmol/L lactic acid, respectively. Whereas, the RS III fractions from cassava starch suspended in water had melt transitions between 158 and 175 °C with low endothermic enthalpies (0.2–1.6 J/g), the thermal transitions of the acid treated samples were indistinct.     

Regrouping in lactating goats increases aggression and decreases milk production

With the aim of testing the hypothesis that regrouping decreases milk production in French Alpine goats that were lactating, a study was done using two groups (n = 8, 7). During their third month after parturition, four goats from each group were exchanged (first regrouping) between pens and left for 2 weeks, then the same two subgroups of four goats were taken back to their original pen for another 2 weeks (second regrouping). In the third regrouping, the two groups were all placed in the one pen. Milk production and social behaviour were measured daily before and after each regrouping. All regroupings led to an increase in aggressive behaviours that last by 1–2 days. Mean daily milk production decreased after first (2.82 ± 0.2 kg versus 2.53 ± 0.2 kg; P < 0.05) but not after second and third regrouping. It is concluded that aggressive behaviour increases after all regroupings, whereas milk production decreases only after the first regrouping, suggesting an important capacity of adaptation to a novel and stressful managements in the French Alpine goat. The study highlights the importance of considering effects of common practices in herd managements on social behaviour and production.     

Genotypic and phenotypic characterization of the dynamics of the lactic acid bacterial population of adjunct-containing Cheddar cheese manufactured from raw and microfiltered pasteurised milk

AIMS: This study investigates the dynamics of the microflora, particularly the lactobacilli, in Cheddar cheese manufactured from raw and microfiltered milk containing different adjunct cultures. METHODS AND RESULTS: Sixteen cheeses - raw milk, adjunct and control cheeses - were manufactured in four trials. Lactobacilli were identified by PCR methods in one trial, and by phenotypic typing for all trials. Numbers of lactobacilli were significantly different at day 1 and 3 months in the control and adjunct-containing cheeses. In the raw milk cheeses, Lactobacillus paracasei was detected throughout ripening, Lact. curvatus at the end, and Lact. plantarum at day 1 only. Lactobacillus strain diversity decreased from raw, control to adjunct cheeses. Enteroccoci and coliform numbers further differentiated raw cheeses from the others. Lactococcal starter numbers also differed in the three cheese types and differences were observed within adjunct cheeses. Although adjunct lactobacilli dominated in the cheese to which they were added, strains with similar phenotypic profiles were also detected on occasions in some of the control cheeses. CONCLUSIONS: The addition of adjunct lactobacilli modified the growth kinetics of both adventitious lactobacilli and starter lactococci during ripening. Appropriate strain tracking is necessary to monitor changes in the population profiles of control and experimental cheeses in trials utilizing adjunct cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigations of the role of adjunct strain(s) in cheeses may be complicated by the interactions between the adjunct and the other cheese strains, and effective strain monitoring by genotypic or phenotypic methods is essential if valid comparisons are to be made.     

Bioethanol production from barley hull using SAA (soaking in aqueous ammonia) pretreatment

Barley hull, a lignocellulosic biomass, was pretreated using aqueous ammonia, to be converted into ethanol. Barley hull was soaked in 15 and 30 wt.% aqueous ammonia at 30, 60, and 75 °C for between 12 h and 11 weeks. This pretreatment method has been known as “soaking in aqueous ammonia” (SAA). Among the tested conditions, the best pretreatment conditions observed were 75 °C, 48 h, 15 wt.% aqueous ammonia and 1:12 of solid:liquid ratio resulting in saccharification yields of 83% for glucan and 63% for xylan with 15 FPU/g-glucan enzyme loading. Pretreatment using 15 wt.% ammonia for 24–72 h at 75 °C removed 50–66% of the original lignin from the solids while it retained 65–76% of the xylan without any glucan loss.

Addition of xylanase along with cellulase resulted in synergetic effect on ethanol production in SSCF (simultaneous saccharification and co-fermentation) using SAA-treated barley hull and recombinant E. coli (KO11). With 3% w/v glucan loading and 4 mL of xylanase enzyme loadings, the SSCF of the SAA treated barley hull resulted 24.1 g/L ethanol concentration at 15 FPU cellulase/g-glucan loading, which corresponds to 89.4% of the maximum theoretical yield based on glucan and xylan.

SEM results indicated that SAA treatment increased surface area and the pore size. It is postulated that these physical changes enhance the enzymatic digestibility in the SAA treated barley hull.     

Evaluation of lamb performance and costs in motherless rearing of German Grey Heath sheep under field conditions using automatic feeding systems

Performance and costs were evaluated for 205 German Grey Heath lambs reared artificially under field conditions using three feeding and three weaning regimes. In phase 1 (pre-weaning), ad libitum feeding of milk replacer (MR) on an automatic lamb feeder was compared to restricted MR feeding on the modified calf feeder “Stand alone II^®” or with buckets. In phase 2 (after weaning at 12 kg body weight), abrupt weaning was compared to gradual weaning systems with reduction of the amount or the concentration of MR over 2 weeks. In phase 1, average daily gains (ADG) were highest in the Ad libitum group (0.262 ± 0.032 kg/d), followed by Bucket and Stand alone with 0.227 ± 0.036 kg/d and 0.209 ± 0.023 kg/d, respectively (P < 0.05). In contrast, feed conversion rate was best for group Stand alone (1.17:1), compared to 1.20:1 in group Bucket and 1.23:1 in group Ad libitum. In phase 2, abrupt weaning caused weight loss in lambs from the Ad libitum and Bucket groups, but not in group Stand alone. No post-weaning growth check was seen in lambs weaned gradually. In general, differences in body weight (BW) between groups were low, and on d 70, no significant difference was seen between feeding groups, whereas lambs weaned abruptly still had lower BW than lambs weaned by reducing the amount of MR (P < 0.05). Total lamb mortality was 3.9%, with no accumulation of infectious disease in any of the feeding or weaning groups. Economic analysis revealed that total costs in phase 1 were highest for group Stand alone (57–70 € per lamb), followed by the Bucket group (63 € per lamb). Although MR consumption was higher in the Ad libitum group, this method was the most cost-efficient (50–54 € per lamb), because purchase price of the feeder and labor costs were relatively low. In phase 2, gradual weaning systems added costs of 4.70–9.50 € per lamb. Our findings indicate that the most efficient way to rear 60–120 lambs artificially under field conditions is by using an automatic ad libitum lamb feeder followed by abrupt weaning.     

Lipase-catalyzed acylation of naringin with palmitic acid in highly concentrated homogeneous solutions

Acylation reactions of naringin with palmitic acid were performed by a lipase after formation of highly concentrated homogeneous solutions. Their initial naringin concentration was 840–950 mM, which is 20–60 times greater than that in organic solvent media. The overall productivity of highly concentrated solutions was more than 15 times greater than those of organic phase media. The addition of DMSO (20–40%, w/w) to substrate mixtures lowered the melting temperature of a naringin–palmitic acid mixture (1:1 molar ratio) to about 40 °C. Reactions at 80 °C apparently followed Michaelis–Menten kinetics despite extremely high substrate concentrations. As the temperature increased from 60 °C to 80 °C, the apparent viscosity of the highly concentrated solution decreased remarkably from 4.31 Pa s to 0.063 Pa s. An activation energy of 7.65 kcal/mol obtained in a range of 60–75 °C suggests a diffusion-control. On the other hand, an activation energy of 17.09 kcal/mol in a range of 75–90 °C indicates a reaction-control. The highest product conversion yield of 33% (mol/mol) was obtained in a 10 h reaction at 80 °C. Addition of activated molecular sieves to the highly concentrated solution increased the product conversion yield by 7% (mol/mol), suggesting that the original equilibrium was disrupted by removing water and then a new equilibrium was reached.     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

Finger temperatures during military field training at 0 to −29 °C

1. Skin and rectal temperatures were recorded continuously in 70 measurements during typical tasks of infantry and artillery training at 0 to −29 °C. The duration of the measurements varied from 55 min to 9.5 h.

2. The distribution of finger skin temperatures was quite similar at ambient temperature ranges 0 to −10 °C and −10 to −20 °C, while at −20 to −30 °C the finger temperatures were clearly lower.

3. At different ambient temperature ranges, 20–69% of finger temperatures were low enough to cause cold thermal sensations.

4. Sensation of cold was experienced at a finger temperature of 11.6±3.7 °C (mean±SD).     

Relationship between male-male and male-female sexual behavior in 5-6-month-old male lambs

To characterize male–male sexual behavior during lamb development, to relate it with lamb body and testicular growth, and with sexual behavior toward estrual ewes, 40 Milchschaf male lambs, weaned at 45 days of age, were kept with ewes that were nursing younger lambs. Experimental lambs were weighed and scrotal circumference was measured every 2 weeks. Male–male sexual behavior was observed during 1–2 h every 2 weeks after birth until 7 months of age. Observations were recorded more intensively (3–4 h on five different days) for 2 weeks (5–6 months of age) as male–male sexual behavior increased during that period. Both mounting and mounted lambs were identified. An individual mounting index (MI) was calculated. To study male–female sexual behavior, lambs were individually located with two estrual ewes, and during 5 min the number of ano-genital sniffing, lateral approaches, mounts, and mounts with ejaculation were recorded. From those data, a libido index was also calculated. Male–male mounts (n = 308) were observed. Courtship behavior was displayed in 25% of interactions; mounts were accepted in 72.1% of attempted mounts. Mounts without previous courtship were accepted more frequently than mounts with previous courtship (P = 0.002). Lamb weight and scrotal circumference were not different according to MI groups. Lambs that mounted more times estrual ewes (first tertile) had greater (P = 0.04) MI (0.61 ± 0.10) than lambs with medium (0.27 ± 0.09) and less (0.30 ± 0.10) MI. The regression between MI and heterosexual libido index was r = 0.33 (P < 0.05). In summary, intensive male–male sexual activity during a short period of male lamb development was observed. There was a positive relationship between sexual behavior of male lambs towards other male lambs and towards estrual ewes.     

Preparation and characterization of solid lipid nanoparticles loaded traditional Chinese medicine

Ultrasonication was employed to prepare solid lipid nanoparticles (SLN). The model traditional Chinese medicine, tetrandrine (TET), was incorporated into SLN. The TET–loaded SLN (TET–SLN) were spherical in the photograph of transmission electron microscope (TEM). The particle size measured by laser diffraction (LD) was found to be 157.3 ± 8.2 nm. Zeta potential analyzer suggested the zeta potential of TET–SLN was −29.36 ± 3.68 mV in distilled water. The entrapment efficiency (EE%) was determined with the sephadex gel chromatogram and high-performance liquid chromatogram (HPLC), and up to 90.59% of TET was incorporated. Stability evaluation showed relatively long-term stability with only slight particle growth (P > 0.05) after storage at room temperature for 4 weeks. Therefore, ultrasonication is demonstrated to be a simple, available and effective method to prepare high quality SLN loaded traditional Chinese medicine.     

Effect of dietary polyunsaturated/saturated fatty acid ratio on heat production and growth performance of chicks under different ambient temperature

Two trials were conducted to investigate the influence of dietary polyunsaturated/saturated fatty acid ratio (P/S) on growth performance and heat production (HP) of chicks under different ambient temperature (Ta). With the exception of Ta, all other treatment processes for two trials were the same. In each trial, 120 Arbor Acres 1-day-old male chicks were allotted to 2 (P/S, 0.6 or 2.4) × 2 (high or low Ta) factorial design with six replicates of five chicks each treatment. Chicks were reared in wire-floored metabolism cages in two temperature-controlled chambers, and were exposed to high Ta (37 and 38 °C at the first day for trial 1 and 2, respectively, and decreased 0.2 °C/day) or low Ta (33 and 28 °C at the first day for trial 1 and 2, respectively, and decreased 0.3 °C/day), for 3 weeks. Chicks were fed ad libitum. Body weight, feed intake and gain:feed were recorded. Excreta were collected for determining metabolizable energy. Energy retention was measured by the comparative slaughter technique; HP was calculated as the difference between metabolizable energy intake and body energy retention.

The results showed that high Ta decreased weight gain (P < 0.01) and feed intake (P < 0.01) in both trials, improved gain:feed in trial 2 (P < 0.01), and decreased HP in trial 2 (P < 0.01) of chicks during 0–3 weeks of age. Increasing dietary P/S did not affect the growth performance and HP of chicks during 0–3 weeks of age. No Ta × dietary P/S interaction among growth performance and HP in chicks was observed.

In summary, increasing dietary P/S did not affect HP, therefore, it is neither detrimental to the growth of chicks reared under high Ta nor is beneficial to the growth of chicks reared under low Ta.     

Aspiration of oocytes from transitional, cycling, and pregnant mares

The aim of this study was to compare the efficacy of three approaches for recovering equine oocytes via transvaginal ultrasound-guided follicular aspiration. Fourteen mares were used as oocyte donors during the spring transition period and physiologic breeding season, and 11 mares were bred for use as oocyte donors during early gestation. In all mares, large (>20 mm) and small (10–20 mm) follicles were aspirated in eight rounds every 10–11 days. In each of the four rounds during the transition period, half the mares received 12.5 mg eFSH once daily for 4 days prior to aspiration. For each of the four rounds during the cycling season, half the mares received 12.5 mg eFSH twice daily for 3 days prior to aspiration. Pregnant mares were aspirated on days 25, 40 and 55 of gestation and received no eFSH. There were more large (>20 mm) follicles in cycling controls (2.25 ± 0.27) and cycling FSH-treated (2.64 ± 0.27) mares than in transitional FSH-treated mares (1.18 ± 0.27). The number of oocytes recovered from small (10–20 mm) follicles varied by mare (P < 0.05) and averaged 1.08 ± 0.22 per aspiration for transitional mares and 1.23 ± 0.22 per aspiration for cycling mares (P > 0.1). The number of oocytes per aspiration from large follicles was greater in cycling FSH-treated mares (0.46 ± 0.09) than in transitional control mares (0.11 ± 0.09). In pregnant mares, more large follicles were present at day 25 than at any other time, and the number of oocytes per aspiration from large follicles was greater at day 25 (0.73 ± 0.16) than at day 55 (0.04 ± 0.18). When compared across all seasons and treatments, the day 25 pregnant mares yielded the greatest number of oocytes per aspiration (2.91 ± 0.66 per mare).