Effect of Temperature and Humidity on Sperm Morphology in Duroc Boars Under Different Housing Systems in Thailand

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1 FULL PAPER Theriogenology Effect of Temperature and Humidity on Sperm Morphology in Duroc Boars Under Different Housing Systems in Thailand Annop SURIYASOMBOON 1,3,5), Nils LUNDEHEIM 2,5), Annop KUNAVONGKRIT 4) and Stig EINARSSON 1,5) 1) Departments of Clinical Sciences, and 2) Animal Breeding and Genetics, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, Uppsala, Sweden, 3) Departments of Animal Husbandry, and 4) Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand and 5) Centre for Reproductive Biology in Uppsala, Uppsala, Sweden (Received 24 June 2004/Accepted 15 April 2005) ABSTRACT. The aim of this study was to investigate the effect of season, temperature, humidity, age of the boar, and semen collection interval on sperm morphology in Duroc boars in Thailand, kept either in a conventional open air system (CONV) or in an evaporative cooling system (EVAP). In total, 1176 ejaculates from 110 sexually mature boars in six CONV herds and five EVAP herds were morphologically examined during a one-year period. Analysis of variance was applied to the data. Minor differences in the sperm morphology traits analyzed were found between the housing systems. There was a significant seasonal effect (two-month periods) on the percentage of morphologically normal spermatozoa (normal1), morphologically normal spermatozoa including spermatozoa with distal cytoplasmic droplets (normal2), proximal cytoplasmic droplets (prox), and sperm head abnormalities (P 0.001). Temperature had a significant effect on normal1 in the CONV system (P 0.01) and in the EVAP system (P 0.05), and on normal2 (P 0.05) and prox (P 0.001) in both systems. Humidity had a significant effect on prox (P 0.05) in the EVAP system. The results also indicate that the sperm morphology traits were mildly affected by the housing systems investigated. A seasonal variation in sperm morphology was found, and high temperatures and high humidity had negative effects on sperm morphology. KEY WORDS: boar, housing system, humidity, sperm morphology, temperature. J. Vet. Med. Sci. 67(8): , 2005 The variation in high ambient temperature and humidity is regarded as an important component causing variation in semen production and semen quality. Several studies have shown that elevated ambient temperature, heat stress and/or hot weather have an adverse effect on semen production [4, 13] and semen quality [2, 7, 8, 10, 12] in boars. In experimental studies, various forms of heat stress have been used to induce these adverse effects. Exposure of mature boars to elevated ambient temperature has been reported to cause alterations in sperm morphology [7, 14]. Additionally, local heating of the scrotum has caused similar disturbances of the spermatogenesis [8, 9]. In most studies, an increased proportion of abnormal spermatozoa has been found after heat treatment, but the results vary among boars, and the results are also related to the different regimes for heat stress that have been used [7 9, 14]. There is apparently a variation among boars in testicular sensitivity to heat. However, no study on the effect of humidity on sperm morphology has, to our knowledge, been reported. There are two housing systems for boars in Thailand: conventional stables (CONV), comprising an open-air system with no walls, and an evaporative system (EVAP), aimed at reducing the temperature by a humidification process. In the EVAP system, water is sprayed onto cooling pads at one end of a closed stable. Hot outdoor air passes through the pads by using an exhaust fan at the other end of building, and the air temperature is cooled down when the water is evaporated. This process reduces the temperature with a complementary increase of the relative humidity in the air [11]. The objective of this study was to investigate the effect of housing system, season, temperature, humidity, age at semen collection, and semen collection interval on sperm morphology in Duroc boars kept in the CONV system and in the EVAP system under tropical conditions in Thailand. MATERIALS AND METHODS Data: This study is based on information collected from 11 commercial pig farms in the central part of Thailand during the period January 2001 until February Six of these farms had the CONV housing system and five farms had the EVAP housing system for the boars. Each farm had only one of these systems. All herds had been operating for at least 10 years, and the EVAP herds had used this system for at least one year before the start of this study. Duroc boars were present in all herds, and the majority of all matings were performed by artificial insemination (AI). In both CONV and EVAP farms, the boars were kept in individual pens (approximately 12 m 2 ), and fed with kg/day of a lactating sow diet containing 16 18% crude protein and 3,100 kcal of digestible energy per kilogram. All boars had free access to water via nipples. The young replacement boars were penned in a quarantine area at least 6 weeks before being used. During this period, the boars were observed for clinical diseases, and were trained for semen collection. During the training period, at least two ejaculates were checked for volume, sperm motility, sperm concentration, and sperm morphology. For a description of the herds (i.e. location, general management) see Suriyasom-

2 778 A. SURIYASOMBOON, N. LUNDEHEIM, A. KUNAVONGKRIT AND S. EINARSSON boon [13]. Temperature and humidity: There are three seasons in Thailand: the hot season from March until June, the rainy season from July until October, and the winter season from November until February. Temperature and humidity were recorded once a day, using a digital Max-Min Thermo- Hygrometer in each of the 11 boar stables. In the CONV boar stables, the device was placed in the middle of the boar stable hanging from a hook m above the floor. In the EVAP boar stables, the device was placed at about onethird of the distance from the fan-end of the building, at the same height as in the CONV stables. Temperature and humidity were recorded daily at about hr, and after each recording, the device was reset to measure a new figure for the next day. The daily recording included maximum, minimum, and actual figure for both temperature and humidity. Sperm morphology examination: The ejaculates were collected using the gloved-hand method, and filtered through gauze to remove the gel fraction. The ejaculates were immediately placed in a water bath at 35 C. The gel-free volume of semen was measured in a beaker. The sperm motility (% progressive) was subjectively estimated under a microscope at 400 magnification. Only ejaculates with more than 60% motility were used for AI. Sperm concentration was assessed with a photometer. Semen was diluted in a preservation medium at 35 C, and transferred into 100 ml insemination bottles. Thereafter, the insemination bottles were cooled down to room temperature for two hours, and then kept at 18 C. Evaluation of sperm morphology was performed for approximately 10 Duroc boars in each herd once a month for a 12-month period. An aliquot from the ejaculate was fixed in 1 ml formol-saline immediately after collection. One drop of semen was also placed on each of 3 4 glass slides, and smears were prepared and air dried. Morphological abnormalities were studied in the formolsaline fixed samples [5] under a phase-contrast microscope at a magnification of 1,000. Altogether 200 spermatozoa were checked in each preparation, and the presence of proximal cytoplasmic droplets, distal cytoplasmic droplets, abnormalities of the midpiece, single bent tails, coiled tails, detached heads, and acrosome defects was recorded. The abnormalities were classified according to the system developed by Bane [1]. For examination of sperm head morphology, the smears were stained with carbolfuchsin-eosin, according to the method described by Williams [15], and modified by Lagerlöf [6]. Altogether, 500 spermatozoa were counted from each ejaculate at a magnification of 1,000 under a light microscope, and the presence of the following sperm head abnormalities was recorded: narrow at the base, pear shaped, abnormal contour, broad-round-giant head, and loose abnormal head. Editing of data: Data were handled by using the SAS program (Ver. 8, SAS Institute Inc., Cary, NC, U.S.A.). There were a total of 1,375 records from sperm morphology examinations available. For each ejaculate, information from formol-saline and smear evaluations was combined with information on system, herd, boar identity, birth date, and collection date, as well as information on semen volume and total sperm production for the actual ejaculate. Incomplete records and records with obvious errors were excluded from the analyses. Some systematic exclusion of records was performed before the statistic analyses: ejaculates with a volume of less than 50 ml or more than 500 ml; ejaculates with a sperm concentration of less than spermatozoa/ml or more than spermatozoa/ml; ejaculates with a total sperm number of less than or more than ; ejaculates where previous collection took place less than three days or more than 14 days before. Also, records from boars younger than 270 days or older than 1,300 days at collection were excluded from the dataset to be analysed. Only boars used for semen collection during at least four months and with at least five examined ejaculates were included in the statistical analyses. After exclusion of records, the analysis comprised 1,176 records, corresponding to 85% of the total number of all morphological records, from 58 and 52 boars in the CONV and in the EVAP systems, respectively. For the statistical analyses, collection month was grouped into 6 2-month periods (January-February, March-April, May-June, July-August, September- October, and November-December), age at collection was grouped into 3 classes ( , , and 750 1,300 days), and previous collection interval was grouped into three classes (3 5, 6 7, and 8 14 days). Twenty-one day moving averages (based on 21 days of information) of daily maximum temperature ( C) and daily minimum relative humidity (% RH) in the boar stables were calculated for each herd and day. Each ejaculate record was merged with its corresponding moving average, with a lag time of 14 days from the end of the 21-day period. If the moving average was based on information from less than 18 days, the moving average was blanked. The moving average of the temperature (T) was grouped into three approximately equally sized classes: T1, T 32 C; T2, 32 C<T 35 C; and T3, T>35 C in the CONV system, and T1, T 29 C; T2, 29 C<T 29.7 C; and T3, T>29.7 C in the EVAP system, respectively. The moving average of humidity (H) was correspondingly grouped into three classes: H1, H 47% RH; H2, 47% RH<H 55% RH; and H3, H>55% RH in the CONV system, and H1, H 68% RH; H2, 68% RH<H 87% RH; and H3, H>87% RH in the EVAP system, respectively. There were 552 and 532 complete records, including both morphology information and the moving averages of temperature and humidity in the CONV and in the EVAP systems, respectively. For frequency analyses, a sub-set of the data above was created, including records on boars with at least three records in each of the three seasons. In this data set, information from 443 records of 37 boars in the CONV system and 453 records of 38 boars in the EVAP system was included. Statistical analyses: Data were statistically analyzed using the SAS program (Ver. 8, SAS Institute Inc., Cary, NC, U.S.A.). Variables analysed were percentages of: morphologically normal spermatozoa (normal1) (100-total % of

3 TEMPERATURE, HUMIDITY, AND SPERM MORPHOLOGY 779 abnormal spermatozoa in formol-saline + total % of abnormal sperm heads in smear); morphologically normal spermatozoa, including spermatozoa with distal cytoplasmic droplets (normal2); spermatozoa with proximal cytoplasmic droplets (prox); spermatozoa with sperm tail defects (sperm tail defects; coiled tail and single bent tail); and spermatozoa with an abnormal sperm head (sperm head defects; narrow at the base, pear shaped, abnormal contour, broad-roundgiant head, and loose abnormal head). To reduce the impact of non-normal distribution, arcsin transformation was applied to the variables normal1 and normal2, and natural log transformation was applied to the last three variables. Results from the analyses were transformed back to ordinary scale in the presentation. Analysis of variance was applied to the data using the General Linear Mixed Model (MIXED) procedure, and two statistical models, including both fixed and random effects, were applied (Table 2). For analysing the seasonal variation, the model included the fixed effects of system, two-month periods, age at collection, previous collection interval, and all possible two-way interactions between them. The random effects of the herd within the system, and the boar within the herd and system were also included in the model. The effects of temperature and humidity were analysed within the systems. The statistical model included the fixed effects of age at collection, previous collection interval, T and H, and all possible twoway interactions between them. The random effects of the herd and boar within the herd were also included in the model. Multiple comparison tests of least-squares means were performed using Bonferroni correction to reduce the risk of getting false significances. The sub-set of data, restricted to boars with at least 3 records in each of the three seasons, was used for studying the performance within boars across season. The approximate best third of the boars (according to the criteria described in Table 3) in the CONV system during the winter season were identified. The same criteria were used to identify the best boars in the EVAP system during the winter season. The proportions of these best boars (during the winter) fulfilling the set criteria during the other two seasons were calculated. Chi-square analyses were performed to study differences in classified distribution of normal1 between seasons within the systems, and between the systems within the seasons. RESULTS Descriptive data: The overall mean values for the variables analysed, after editing, are shown in Table 1. The mean volume of the ejaculates in the CONV herds (229 ml) was higher than in the EVAP herds (203 ml), but sperm concentration in the CONV herds ( ) was lower than in the EVAP herds ( ). Additionally, the total sperm number per ejaculate in the CONV herds was higher than in the EVAP herds. The previous semen collection interval and age at collection were similar for both systems; 7.1 days and 698 days in the CONV system, and 6.4 days and 626 days in the EVAP system, respectively. There was a higher moving average of maximum temperature and a higher cor- Table 1. Descriptive statistics for variables included in the analyses System N Mean SD Range Ejaculate volume (ml) CONV EVAP Sperm concentration ( 10 6 /ml) CONV EVAP Total sperm number per ejaculate ( 10 9 ) CONV EVAP Collection interval (days) CONV EVAP Age at collection (days) CONV EVAP Maximum temperature, moving average ( C) CONV EVAP Minimum humidity, moving average (% RH) CONV EVAP Percentage of spermatozoa with - normal morphology (normal1) CONV EVAP normal morphology including distal cytoplasmic droplets CONV (normal2) EVAP proximal cytoplasmic droplets (prox) CONV EVAP distal cytoplasmic droplets CONV EVAP sperm tail defects CONV EVAP sperm head defects CONV EVAP

4 780 A. SURIYASOMBOON, N. LUNDEHEIM, A. KUNAVONGKRIT AND S. EINARSSON Table 2. The levels of significance for the effects included in the statistical models Analyses including both systems Analyses within system Fixed effects Normal1 a) Normal2 b) Prox c) Sperm tail Sperm head Normal1 a) Normal2 b) Prox c) Sperm tail Sperm head and interactions defects defects defects defects CONV EVAP CONV EVAP CONV EVAP CONV EVAP CONV EVAP System ns (d) ns ns ns ns Two month period *** *** *** ns *** Age at collection ns ns ns * ns ns ns ns ns ns ns * ns ns ns Collection interval ns ns ns ns ns ns ns ns ns ** ns ns ns ns ns Temperature ** * * * *** *** ns ns ns ns Humidity ns ns ns ns ns * ns ns ns ns Interaction between : System and two month period ns ns * ns ns System and collection interval ns ns ns ns ns System and age at collection ns ns ns ns ns Two month period and age at collection * ns * ns ns Two month period and collection interval ns ns ** ns * Age at collection and collction interval ns ns ns ns ns ns ns * * ns ns ns ns ** ns Temperature and age at collection ns ns ns ns ns ns ns ns ns ns Temperature and collection interval ns ns ns ns ns ns ns ns ns ns Humidity and age at collection * ns * ns ns ns ns * ns * Humidity and collection interval ns ns ns ns ns ns ns ns * ns Temperature and humidity ns * * ** ns ns ns ns * ** Number of observations (a) Morphologically normal spermatozoa. (b) Morphologically normal spermatozoa including spermatozoa with distal cytoplasmic droplets. (c) Spermatozoa with proximal cytoplasmic droplets. Fig. 1. Seasonal variation in sperm morphology traits (least squares-means for each two month period). Values with one letter in common are not significantly different (P>0.05). responding standard deviation (SD) in the CONV system compared with the EVAP system. The corresponding ranges were 27.3 C 40.0 C and 29% RH 78% RH in the CONV system, and 23.2 C 31.6 C and 43% RH 96% RH in the EVAP system, respectively. However, the moving average of the minimum humidity and its SD was lower in the CONV than in the EVAP system. The sperm quality was generally good. No major differences between the morphology variables in the two systems could be seen: percentages of normal1, prox, and sperm head defects were approximately 80%, 4%, and 7% in the CONV system, and 81%, 4%, and 7% in the EVAP system, respectively. The levels of significance for all fixed effects included in the statistical models are presented in Table 2. When analysing the two systems together, no significant effect of system and collection interval was found on any of the variables analysed. There was a significant effect of twomonth periods on all variables (P 0.001), except on sperm tail defects. There was a significant interaction between two-month periods and collection interval for prox (P 0.01). There was a significant decrease in the proportion of sperm tail defects when age at collection increased (P 0.05) (least-squares means: 0.73% [young]; 0.68% [middle]; 0.44% [old]). Also, there was a significant increase in prox (P 0.01) when collection interval increased in the CONV system (least-squares means: 0.70% [3 5 days]; 0.85% [6 7 days]; 1.55% [8 14 days]). Effect of season: Figure 1 shows the seasonal variation in the analysed variables. Both normal1 and normal2 decreased from January/February to March/April, increased gradually to May/June, after which they decreased to July/ August, and thereafter gradually increased to the same level as in January/February. Prox increased from January/February to May/June, decreased thereafter gradually to

5 TEMPERATURE, HUMIDITY, AND SPERM MORPHOLOGY 781 Fig. 2. The effect of temperature and humidity in sperm morphology traits (least squares-means) in the CONV (-----) and EVAP ( ) systems. Values with one letter in common are not significantly different (P>0.05). Average temperature/humidity for each class is given within brackets. November/December. Sperm head defects increased from January/February to March/April, decreased thereafter to May/June, gradually increased again to September/ October, and decreased again to November/December. Normal1 and normal2 were lowest in July/August, while prox and sperm head defects were highest in May/June and September/October, respectively. Effect of temperature and humidity: The average daily maximum temperatures per T-class for the CONV system were T1: 30.6 C, T2: 33.6 C, and T3: 36.2 C. Corresponding temperatures for the EVAP system were T1: 28.1 C, T2: 29.4 C, and T3: 30.2 C. The corresponding averages of the daily minimum humidity per H-class for the CONV system were H1: 42% RH, H2: 51% RH, and H3: 63% RH, and for the EVAP system they were H1: 59% RH, H2: 75% RH, and H3: 92% RH. There was a significant effect of T on normal1, normal2, and prox in both systems (Table 2). A significant effect of H on prox (P 0.05) was also found in the EVAP system (Table 2). Figure 2 shows the effect of temperature and humidity on sperm morphology. There was a significant decrease in normal1 (P 0.01), and a significant increase in prox (P 0.001) when temperature increased from T1 to T2 (on average, 30.6 C to 33.6 C) in the CONV system. In the EVAP system, there was a significant decrease in normal1 (P 0.05) when temperature increased from T1 to T3 (on average, 28.1 C to 30.2 C), and a significant increase in prox (P 0.001) when temperature increased from T1 to T2 (on average, 28.1 C and 29.4 C). There was a significant increase in prox (P 0.05) when humidity increased from H2 to H3 (on average, 75% RH to 92% RH) in the EVAP system (least-squares means: 1.02% and 1.75%). Combined effect of temperature and humidity: Figure 3 shows the combined effect of temperature and humidity on the sperm morphology variables analysed. The interaction effects were, in most cases, not significant or weakly significant, and the combined effects were, in most cases, just describing the independent effects of T and H. In the EVAP system, where the interaction between T and H was significant (P 0.01) for both normal2 and sperm head defects (Table 2), the combination of T3 and H3 seems to be most negative, and high humidity (H3) in combination with low

6 782 A. SURIYASOMBOON, N. LUNDEHEIM, A. KUNAVONGKRIT AND S. EINARSSON Fig. 3. The combined effect of temperature and humidity on sperm morphology traits within the systems (least-squares means). or medium temperature seems to be positive for the production of normal spermatozoa. Distribution of boars: The approximate best third of the boars in the CONV system, according to the different criteria given in Table 3, were identified during the winter season. The same criteria were also applied to the boars in the EVAP system. Using these criteria, only 3 out of 38 boars in the EVAP system had high volume during the winter season, compared with 14 out of 37 boars in the CONV system. A larger number of the best boars in the CONV system (9

7 TEMPERATURE, HUMIDITY, AND SPERM MORPHOLOGY 783 Table 3. Number of boars fullfilling the specified criteria of being best during winter season, as well as during the hot and rainy seasons CONV EVAP Winter Hot Rainy Winter Hot Rainy Parameter season season season season season season Ejaculate volume high 14/37 5/14 3/14 3/38 3/3 2/3 ( 270 ml) (37.8%) (7.9%) Total sperm number high 13/37 9/13 8/13 9/38 2/9 2/9 per ejaculate ( ) (35.1%) (23.7%) Normal1 high 11/37 3/11 0/11 7/38 0/7 0/7 ( 90%) (30.0%) (18.4%) Normal2 high 15/37 7/15 2/15 14/38 2/14 0/14 ( 91%) (40.5%) (36.8%) Prox low 14/37 3/14 2/14 9/38 2/9 2/9 ( 0.9%) (37.8%) (23.7%) Sperm tail defects low 12/37 4/12 2/12 11/38 3/11 3/11 ( 0.6%) (31.6%) (28.9%) Sperm head defects low 13/37 5/13 1/13 15/38 0/15 0/15 ( 4.2%) (35.1%) (39.5%) Table 4. Distribution of classified normal1 within systems and seasons, number of records, and percentage within the systems and seasons CONV Percentage of normal1 Winter Hot Rainy Total Winter Hot Rainy Total 95 % (6.8%) (7.8%) (4.1%) (6.3%) (10.9%) (6.2%) (6.5%) (7.9%) EVAP 90% < 95% (32.9%) (20.4%) (14.4%) (22.7%) (25.4%) (20.2%) (19.7%) (21.8%) 85% < 90% (24.1%) (18.9%) (21.1%) (21.4%) (20.7%) (17.6%) (15.9%) (18.1%) 80% < 85% (12.6%) (15.0%) (12.4%) (13.4%) (15.0%) (14.5%) (20.8%) (16.7%) 70% < 80% (15.0%) (16.0%) (18.1%) (16.3%) (16.1%) (20.2%) (18.6%) (18.3%) 60% < 70% (4.3%) (11.7%) (10.8%) (8.9%) (6.2%) (10.9%) (7.6%) (8.2%) < 60% (4.3%) (10.2%) (19.1%) (11.0%) (5.7%) (10.4%) (10.9%) (9.0%) Number of records Normal1 - Means Normal1 - SD Normal1 - Range /13) than in the EVAP system (2/9) also had a high total number of spermatozoa per ejaculate during the hot and rainy seasons. For the other variables analysed, approximately the same portion of boars in the CONV system as in the EVAP system fulfilled the set criteria during the winter season. Only a few (or sometimes no) boars that had good sperm quality during the winter season continued to have good sperm quality (high percentage of normal spermatozoa

8 784 A. SURIYASOMBOON, N. LUNDEHEIM, A. KUNAVONGKRIT AND S. EINARSSON or low percentage of sperm defects) also during the hot and rainy seasons. Distribution of normal1: The proportion of ejaculates with 90% normal1 was approximately 30% in both systems, being higher during the winter season than during the hot and rainy seasons (Table 4). In addition, the corresponding proportion of records with < 80% normal1 was the same (36%) in both systems, and lowest during the winter season (approximately 24% and 28% for CONV and EVAP systems, respectively). Chi-square analyses show a seasonal variation in normal1 (classified) in the CONV system (P 0.001), but not in the EVAP system (P=0.188). Furthermore, there was a significant difference between the two systems in normal1 (classified) during rainy season (P 0.05), but not during the winter or hot season. During the rainy season, there was a higher proportion of records with < 60% normal1 in the CONV system (19.1%), compared with the EVAP system (10.9%). DISCUSSION The present study investigated the effect on sperm morphology in Duroc boars under tropical climatic conditions of two housing systems, CONV and EVAP, the influence of season, the effect of temperature and humidity, age at collection, and collection interval. The same criteria for editing of data were used as in our previous study [13], except for the upper age of the boars at collection, which was extended from 990 days to 1,300 days. This resulted in 84 more records being included in the analyses. The present results show no significant effect of system on sperm morphology. No effects of age of boar or collection interval on sperm morphology, except for sperm tail defects and prox, respectively, were found. Therefore, the discussion is mainly focused on the seasonal, temperature, and humidity effects on sperm morphology. In Thailand, the day-length is almost equal throughout the year, approximately 12 ± 1 hr. Thus, the seasonal variation in sperm morphology in this study can be regarded to be caused mainly by the variation in temperature and humidity. The overall results of the present study reveal that not only elevated temperature, but also elevated humidity has a negative impact on sperm morphology. The maximum temperature and minimum humidity were chosen as microclimatic indicators when analysing the climatic effect on sperm morphology because the maximum temperature always occurred contemporarily with the minimum humidity during early afternoon. From a human point of view, this time of the day is felt to be the hottest period. The present comprehensive investigation of sperm morphology of Duroc boars, kept in CONV or EVAP systems, reveals low percentages of sperm defects/abnormalities in the majority of examined ejaculates. Moreover, the percentages of normal1 in these boars, kept in a tropical climate with high temperature and high humidity, are approximately at the same level as for Duroc boars kept in temperate areas (Wallgren and Lundeheim, Uppsala, Sweden, unpublished results). Seasonal variation: This study shows a fluctuation in sperm quality parameters during the year (Fig. 1). A reduction in normal1 and normal2 could be seen during the first part of the hot season, and during the first part of the rainy season. Also, prox and sperm head defects were elevated at the same time. This might be related to the variation in microclimate, with an increased temperature during the hot season, and an increased humidity during the rainy season [13]. The recovery of normal1 and normal2 after the hot and rainy seasons was a gradual process. The reason for this gradual recovery, followed by a gradual impairment, might be the slow change to lower temperature during the rainy season, compared with the hot season, and the lower temperature and the lower humidity during the winter season, compared with the rainy season. The total sperm production and normal1 were higher during the winter season than during the other seasons. The present results show that there is a variation between boars in sperm production and sperm morphology over the year (Table 3). This variation is most likely due to differences in ability among the boars to adapt to the changes in temperature and humidity. Only a few boars had high sperm production and high sperm quality over all three seasons. These boars seem to be more resistant to climatic changes in the environment. Thus, the majority of boars seem to be sensitive to a harsh climate, and had fluctuations in sperm production and sperm morphology. This observation is in agreement with results from some earlier experimental studies showing an individual variation in sensitivity among boars to elevated ambient temperature and local heating of the scrotum [2, 7, 9]. The distribution of normal1 (classified < 60%) was higher during the rainy season in the CONV system than in the EVAP system (Table 4). During the rainy season, the average maximum temperature was higher in the CONV system than in the EVAP system (approximately 4 C), while the average minimum humidity was higher in the EVAP system than in the CONV system (approximately 30% RH) [13]. One explanation might therefore be that the higher temperature in the CONV system was more negative than the higher humidity in the EVAP system for testicular function during the rainy season. The present results show a seasonal variation in distribution of normal1 (classified) in the CONV system. This variation is most likely due to the higher temperature in the CONV system than in the EVAP system over the year [13]. Temperature and humidity: The present results indicate a clear effect of a 21-day moving average of maximum temperature on sperm quality (normal1, normal2, and prox) in both the CONV and EVAP systems (Figs. 2 and 3). In the CONV system, there was a higher variation (SD) in normal1 than in the EVAP system. This might be explained by the higher variation in temperature in the CONV system than in the EVAP system. An average temperature higher than T1 (on average 30.6 C) in the CONV system, and higher than T1 (on average 28.1 C) in the EVAP system decreased both

9 TEMPERATURE, HUMIDITY, AND SPERM MORPHOLOGY 785 normal1 and normal2, and increased prox. The gradual impairment of sperm quality (normal1, normal2, and prox) at higher ambient temperatures is in agreement with the results of several previous experimental studies; elevated temperatures in hot chamber conditions [2, 3, 7, 10, 12] and scrotum insulation [8, 9]. The impaired sperm morphology is considered to be due to an alteration in the seminiferous epithelium caused by elevated temperature [9]. The high humidity had a significant negative effect on prox, but not on normal1 and normal2 in the EVAP system. High temperature combined with high humidity in the EVAP system, decreased both normal1 and normal2, and increased prox. One might speculate that a combination of elevated temperature and humidity is more deleterious for testicular function (sperm morphology) than high temperature and high humidity separately. Moreover, increased incidences of proximal droplets mostly depend on testicular disturbances. However, experimental elevation of temperature has shown some alterations of the sperm morphology as early as 8 days after the end of 100 hr exposure to 35 C [7], indicating that the epididymal influence on sperm maturation was disturbed. Similar results were obtained by Malmgren [8]. Therefore, an increase of prox in the present study might also indicate a disturbed epididymal function. Furthermore, the boars had similar sperm morphology patterns in both systems. It may be assumed that the boars exposed to either high temperature and low humidity in the CONV system or low temperature and high humidity in the EVAP system reacted in a similar way. Further investigations, specifically on the effect of humidity on sperm morphology, are needed. It can be concluded that only minor differences in the sperm morphology traits analyzed were found between the housing systems. A seasonal variation in sperm morphology was found. There was a negative effect of high temperature and high humidity on sperm morphology. ACKNOWLEDGEMENTS. The Royal Thai Government and Chulalongkorn University, Thailand, are acknowledged for granting A. Suriyasomboon study leave. The authors thank Mr. Chinda Singh-lor for his examination of sperm morphology in this study. REFERENCES 1. Bane, A Acrosomal abnormality associated with sterility in boar. pp In: Proc. 4 th Int. Congr. Anim. Reprod. & A.I., The Hague, IV. 2. Cameron, R.D. and Blackshaw, A.W The effect of elevated ambient temperature on spermatogenesis in the boar. J. Reprod. Fertil. 59: Christenson, R.K., Teague, H.S., Grifo A.P. Jr. and Roller, W.L The effect of high environmental temperature on the boar. pp In: Ohio Swine Research Information Report: Research Summary 61. Ohio Agric. Res. Dev. Center, Wooster. 4. Colenbrander, B., Feitsma, H. and Grooten, H.J Optimizing semen production for artificial insemination in swine. J. Reprod. Fertil. (Suppl.) 48: Hancock, J The morphology of boar spermatozoa. J. R. Microsc. Soc. 76: Lagerlöf, N Morphologische Untersuchungen über Veränderungen im Spermabild und in den Hoden bei Bullen mit verminderter oder aufgehobener Fertilität (Research concerning morphologic changes in the semen picture and in the testicles of sterile and subfertile bulls) Acta Pathol. Microbiol. Scand. Suppl. 19, Thesis, Stockholm. 7. Larsson, K. and Einarsson, S Seminal changes in boars after heat stress. Acta Vet. Scand. 25: Malmgren, L Experimentally induced testicular alterations in boars: sperm morphology changes in mature and peripubertal boars. Zbl. Vet. Med. A 36: Malmgren, L. and Larsson, K Experimental induced testicular alterations in boars: histological and ultrastructure finding. Zbl. Vet. Med. A 36: McNitt, J.I. and First, N.L Effects of 72-hour heat stress on semen quality in boars. Int. J. Biometeorol. 14: Simmons, J.D. and Lott, B.D Evaporative cooling performance resulting from changes in water temperature. Appl. Eng. Agric. 12: Stone, B.A Heat induced infertility of boars: The interrelationship between depressed sperm output and fertility and an estimation of the critical air temperature above which sperm output is impaired. Anim. Reprod. Sci. 4: Suriyasomboon, A., Kunavongkrit, A., Lundeheim, N. and Einarsson, S Effect of temperature and humidity on sperm production in Duroc boars under different housing systems in Thailand. Livest. Prod. Sci. 89: Wettemann, R.P., Wells, M.E., Omtvedt, I.T., Pope, C.E. and Turman, E.J Influence of elevated ambient temperature on reproductive performance of boars. J. Anim. Sci. 42: Williams, W.W Technique of collecting semen for laboratory examination with a review of several diseased bulls. Cornell Vet. 10: