Influence of breed and season on lactoperoxidase system components in cow's milk in Western Cameroon

F A Fonteh

Department of Animal Production, Faculty of Agriculture, University of Dschang.
P.O. Box 447 Dschang-Cameroon.
fontehflorence@yahoo.com

Abstract

A study was carried out in the western part of Cameroon to assess the levels of lactoperoxidase activity and thiocyanate in the milk of different cow breeds. 27 herds were identified and 54 milking cows selected.

Enzyme activity was significantly higher (P < 0.05) in the Goudali breed (1859 µM product/min) than in the other breeds. The greatest variation was observed among Red Fulani cows (1268 to 2241 µM product/min.). Thiocyanate concentration was highest (P < 0.05) in the Goudali breed (13.60 ppm) and also the most varied (8.88 to 19.8ppm). Enzyme activity and thiocyanate content were significantly higher in the dry than in the rainy season, irrespective of the sampling site and the year of sampling.

Such significant variations indicate that the efficiency of the lactoperoxidase system in preserving milk will also vary. Therefore a preliminary investigation is recommended for each area of interest so that the concentrations of thiocyanate naturally present in the local milk are known. This will serve as a basis in determining the quantity that needs to be further added towards optimising the system.

Key words: Breed, Cameroon, cows, lactoperoxidase activity, milk preservation, thiocyanate.

Introduction

As in most countries in Sub-Saharan Africa, the dairy sector in Cameroon is still underdeveloped. Most of the milk is produced by local breeds which are also destined for meat production. Cattle account for 16% of national agricultural production and are mainly found in the western and northern parts of the country (Kameni et al 1999). The predominant breeds include the Zebus (White Fulani and Red Fulani), Akou, Nd'ama and Goudali which all belong to the Bos indicus family. The genetic performance of these breeds is quite low (Atekwana and Maximuangu 1981; Mrode 1988) and the milk produced does not satisfy the demand, this deficit being met by the importation of milk and milk products (Tambi 1991; Kameni et al 1999). Attempts have been made to increase production by the introduction of exotic breeds (such as the Brown Swiss, Montbeliard, Holstein-Friesian and Jersey) which are used either for cross breeding with the local breeds or as pure breeds for milk production (Tambi 1991; Njwe 1999). Despite these attempts, per caput consumption still exceeds the per caput domestic production. In addition to poor performance, other constraints to dairy production that have been identified include high cost of production, processing and transportation; lack of an organised milk distribution and marketing system, poor incentives to farmers and inadequate infrastructure facilities particularly electricity, which render the preservation of raw milk almost impossible in rural areas (Tambi 1991; Fonteh 2001).

Due to poor storage facilities, a lot of the milk is lost through spoilage during the rainy season when production is at its peak. This preservation problem can be alleviated by the application of the lactoperoxidase system (LPS). The system comprises the enzyme lactoperoxidase (LP), and two substrates: thiocyanate and hydrogen peroxide. These substrates are naturally present in milk but in limited quantities thereby reducing the efficacy of the LPS (Reiter and Harnulv 1984). To make the LPS more efficient, variable quantities of thiocyanate and peroxide must be added to the milk. Trials in many developing countries have shown that in the absence of refrigeration, the shelf life the raw milk can be significantly prolonged by the application of the LPS (Harnluv and Kandasamy 1982; Kamau and Kroger 1984; Kumar and Mathur 1989; Ridley and Shalo 1990; El-Agamy et al 1992). These workers also demonstrated that the efficiency of the system (which is generally assessed by determining enzyme activity) varies with the amount of substrates present. Although the IDF (1994) recommends that 10 ppm of thiocyanate and 10 ppm of peroxide be used to render the LPS effective, Kamau and Kroger (1984) as well as Ridley and Shalo (1990) have recommended that for each specific area, the optimal dosages of the substrates must first be determined before the LPS is applied.

Other studies have revealed that the level of the different components of the LPS as they occur naturally in milk vary significantly depending on several factors mainly the breed, feed and stage of lactation (Zapico et al 1991; Saad de Schoos et al 1999; Althaus et al 2001; Fonteh et al 2002). Therefore, baseline studies have to be made in each area, to determine the concentration of thiocyanate naturally present in the local milk before recommendations can be made as to the quantity of thiocyanate needed to be added so as to optimise the LPS. This has not yet been done in the Cameroonian context, hence the purpose of the present study.

Materials and methods

Study area and farms

Data were collected from two main areas: Dschang in the West Province and Bamenda in the North West Province. These provinces make up the Western highlands of Cameroon. This area falls within the sudano-guinean zone (latitude 5-7^oN, longitude 8-12^oE). The average annual temperature varies between 16 and 27^oC while the relative humidity is 40-97%. There are two main seasons: the rainy season (mid March to October) and the dry season (November to early March) and the mean annual rainfall is about 2000 mm (Teguia et al 1997).

The criteria used for selecting farms in the Dschang area included accessibility and number of lactating cows in the herd. Since the cows in this area graze freely on natural pastures, any similarities between farms were minimised by ensuring that the inter-farm distances were at least 1 km, and that no two farms selected were located on the same hill. The distance between farms in Bamenda was not taken into consideration because the feeding style is zero grazing. A total of 5 villages and 27 farms (9 from the Dschang area and 18 from the Bamenda area) were identified for data collection (Table 1).

Table 1. Farms and cows selected for data collection
Area	Name of Village	Number of farms	Number milking cows selected
Area	Name of Village	Number of farms	Red Fulan	Goudali	Holstein
Dschang
	Fongo-Tongo	4	9	7	0
	Fotomena	5	12	6	0
Bamenda
	Nkwen	5	0	2	4
	Njimbi	5	0	0	5
	Bambui	8	2	1	6
Total	5	27	23	16	15

Selection of breeds and cows

One exotic (Holstein-Friesian) and two indigenous breeds (Goudali and Red Fulani) were selected for the study since these are the most predominant in the study area. All cows selected were in their mid-stage of lactation (between 3 and 4 months after calving) and at their second calving. Individual milk was collected from 54 cows (23 Red Fulani, 16 Goudali and 15 Holstein), transported under ice and analysed within 10 hours after collection for LP activity and thiocyanate levels. Sampling and analysis were repeated two weeks later. This was in the month of September.

For studies on seasonal effects, bulk milk was obtained from two farms per village and mixed together. Sampling was done four times per farm and each collection was within 2 weeks' interval of the other. All samples were transported under ice and analysed for enzyme activity and thiocyanate concentration not more than 10 hours after sampling. Collection and analysis were done in the rainy season (July/August) and the procedure repeated during the dry season (January/February) over three consecutive years.

Chemical and statistical analyses

Thiocyanate concentration was determined in duplicate according to Marshall et al (1986): milk samples were first deproteinised by mixing 2ml of 20% trichloroacetic acid with 4ml of milk. The supernatant fluid (1.5ml) was mixed with 1.5ml of ferric nitrate dye and the absorbance measured immediately at 460nm and compared with a standard curve. Lactoperoxidase activity was determined according to the modified method of Pruitt and Kamau (1994). In this procedure, 0.1ml of milk was mixed with 2ml of 1mM 2,2' azino-di-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) in phosphate buffer (0.1M, pH 6.7) in a cuvette. The reaction was initiated by adding 1ml of 0.3mM hydrogen peroxide to the mixture. The reaction was monitored for 1 minute and the absorbance recorded at 412nm and the results are expressed as µM of product per minute. Each sample was analysed in five replicates and standard deviation between replicates was less than 5%.

Data collected were subjected to analysis of variance and significant means separated using Duncan's multiple range test (Steel and Torrie 1980). For seasonal effects, the student t-test was used and significant differences between the two seasons of each year were evaluated at 95% confidence interval (Steel and Torrie 1980).

Results

Lactoperoxidase activity in different breeds

LP activity was highest (1859 ± 42 µM product/min.) in the Goudali breed and lowest (1364 ± 28 µM product/min.) in the Holstein-Friesian breed (Table 2). The range in enzyme activity was quite wide within breeds. The widest variation was recorded in the cows of Red Fulani breed, where values ranged from 1269 to 2241 µM product/min. Consequently, the standard deviation from the mean was very high (379 µM product/min.). In fact very large differences were observed in some cases between samples from the same cow although the sampling interval was only two weeks. The least variation was observed in cows of the Holstein-Friesian breed where enzyme activity values ranged from 941to 1690 µM product/min. and the standard deviation was 156 µM product/min. LP activity was not significantly different between the Red Fulani and the Goudali breeds. However, enzyme activity was significantly higher (P < 0.05) when these two breeds were separately compared to the Holstein-Friesian breed.

Table 2. Lactoperoxidase activity (mM product/min.) and thiocyanate concentration (ppm) in various breeds of cow
Breed of cow	Total number farms visited	Total number of cows sampled	Enzyme activity (mM product/min.)		Thiocyanate concentration (ppm)
Breed of cow	Total number farms visited	Total number of cows sampled	Range (standard deviation)	Mean ± standard error	Range (standard deviation)	Mean ± standard error
Holstein-Friesian	15	15	941- 1690 (156)	1364^a ± 28	6.45- 12.86 (1.95)	8.71^a ± 0.36
Red Fulani	6	23	1269 – 2241 (379)	1765^b ± 56	7.85 -15.15 (2.66)	11.38^b ± 0.49
Goudali	6	16	1488 – 2339 (237)	1859^c ± 42	8.88 - 19.77 (3.67)	13.60^c ± 0.65
a, b, c (means ± standard error) bearing different superscripts within the same column differ at P <0.05

Thiocyanate concentration in different breeds

The highest value for thiocyanate concentration was observed in the Goudali breed (13.6 ppm) while the lowest was observed in the Holstein-Friesian breed (8.31 ppm). Variations within the same breed were also quite high. The greatest variation within breed was observed among the Goudali cows where values ranged from 8.88 to 19.77 ppm. Consequently, the standard deviation from the mean was also highest in this breed (3.67 ppm). The least variation was observed in the Holstein-Friesian breed (6.45 - 12.86 ppm) with a standard deviation of 1.95 ppm. Statistical analyses revealed that the thiocyanate levels were significantly different (P < 0.05) between all the three breeds.

Effect of season on thiocyanate concentration

Irrespective of the collection site, the thiocyanate concentration was higher during the dry season (Table 3). The greatest concentration was observed in the milk collected from Fongo-Tongo during the dry season of the second year (16.1 ± 0.67 ppm) while the lowest was found in the milk from Njimbi collected during the rainy season of the second year (8.94 ± 0.32 ppm). Throughout the study period, the greatest variations in thiocyanate levels were noticed in samples from Fotomena while the least variations were found in samples from Njimbi. There was a continuous rise in thiocyanate content in the milk from Nkwen from one dry season to the other. Although thiocyanate content was consistently higher in the dry season during the three years of sampling for the other collection villages, no definite trend was evident. Statistical analyses revealed that irrespective of the year of sampling, the thiocyanate content was significantly higher (95% CI) in the dry season than in the rainy season in every collection village.

Table 3. Seasonal variations in thiocyanate concentration (ppm) in bulk milk over three consecutive years
Collection village	Year 1		Year 2		Year 3
Collection village	Rainy	Dry	Rainy	Dry	Rainy	Dry
Fongo-Tongo	13.63^a ± 0.63	14.94^b ± 0.50	12.56^a ± 0.43	16.13^b ± 0.67	13.31^a ± 0.52	15.94^b ± 0.63
Fotomena	12.19^a ± 0.63	15.81^b ± 0.79	12.63^a ± 0.44	14.94^b ± 0.75	13.38^a ± 0.40	15.56^b ± 0.71
Njimbi	9.25^a ± 0.25	11.88^b ± 0.25	8.94^a ± 0.32	10.13^b ± 0.37	9.44^a ± 0.27	12.56^b ± 0.41
Bambui	10.75^a ± 0.28	12.00^b ± 0.52	11.06^a ± 0.37	13.75^b ± 0.34	10.81^a ± 0.36	13.06^b ± 0.29
Nkwen	9.75^a ± 0.31	11.94^b ± 0.42	10.19^a ± 0.36	12.38^b ± 0.42	10.13^a ± 0.32	13.63^b ± 0.54
a, b means (± standard error) bearing different superscripts within the same row for each year differ at P <0.0

Effect of season on enzyme activity

Greatest enzyme activity was recorded in Fongo-Tongo during the dry season of the second year (2321 ± 85 µM product/min) while the lowest activity was found in the sample from Njimbi during rainy season of the first year (1807 ± 30 µM product/min.) (Table 4). Generally, variations in enzyme activity were more pronounced in samples collected during the rainy season, as reflected by their relatively higher standard error values. The greatest variation in enzyme activity was observed in samples from Fotomena during the rainy season of the first year with a standard error of 109 µM product/min. The lowest variation was observed in samples collected from Njimbi during the dry season of second year whose standard error was 28 µM product/min. A continuous yearly increase in enzyme activity was observed in the samples from Njimbi in both the rainy and dry seasons. In Bambui village, a yearly increase was observed only in the dry seasons. No definite trend emerged during the study period for the other collection villages. In all cases, enzyme activity was significantly higher (95% CI) in the dry than in the rainy season during each year of study.

Table 4. Seasonal variations in lactoperoxidase activity (µM Product/min.) in bulk milk over three consecutive years
Collection village	Year 1		Year 2		Year 3
Collection village	Rainy	Dry	Rainy	Dry	Rainy	Dry
Fongo-Tongo	1787^a ± 84	2279^b ± 76	1844^a ± 72	2321^b ± 85	1806^a ± 59	2271^b ± 50
Fotomena	1692^a ± 109	2282^b ± 87	1555^a ± 75	2102^b ± 69	1585^a ± 93	2179^b ± 88
Njimbi	1108^a ± 30	1378^b ± 39	1184^a ± 33	1462^b ± 28	1210^a ± 34	1481^b ± 32
Bambui	1258^a ± 50	1532^b ± 33	1316^a ± 32	1582^b ± 38	1276^a ± 35	1607^b ± 33
Nkwen	1330^a ± 35	1634^b ± 45	1366^a ± 39	1615^b ± 37	1230^a ± 34	1688^b ± 36
a, b means (± standard error) bearing different superscripts within the same row for each year differ at P <0.0

Discussion

The variations in lactoperoxidase activity and thiocyanate concentration observed in this study were wide indeed but not unusual. Medina et al (1989) observed similar trends while studying ewe's milk. Zapico et al (1990) reported variations in milk from individual goats that ranged from 0.67 to 11.2 ppm. In yet other studies, Zapico et al (1991) and Saad de Schoos et al (1999) reported significant differences in both LP activity and thiocyanate content within and between different breeds of goat. Fonteh et al (2002) reported similar variations within and between goats and Holstein-Friesian dairy cows throughout a lactation period.

The feed composition is arguably the most important factor that influences the thiocyanate levels in dairy cattle (Bjorck et al 1975; Reiter and Harnluv 1984). Higher thiocyanate values found in the milk from the indigenous breeds (Red Fulani and Goudali) when compared to the Holstein-Friesian breed were most probably as a result of differences in the chemical composition of their feeds. It is possible that the kinds of natural grasses that thrive in this region are high in glucosinolates and/or cyanogenic glucosides (Dahlberg et al 1984), which are the major dietary sources of thiocyanate in animal feed. The thiocyanate level in the milk from cows grazing on natural pastures is reportedly higher than that from non-grazing cows (Reiter and Harnluv 1984; Ekstrand 1994). It will be worthwhile in future to investigate the thiocyanate levels in the predominant grasses in these regions. The higher variability in thiocyanate levels in the indigenous breeds reflects the degree of diversity of the grasses present in the grazing land and the individual ability of the cows to selectively browse on particular grass types. Since all the Holstein-Friesian cows are fed the same diet, variability in thiocyanate levels are minimised. Furthermore, differences in thiocyanate levels between breeds could be due to genetic factors. Some breeds tend to be more efficient in digesting and metabolising glucosinolates and/or cyanogenic glucosides than others (Dahlberg et al 1984; Zapico et al 1991). This may account for the relatively higher levels observed in the milk from the Goudali breed when compared to that from the Red Fulani breed although both were under the same feeding regime. Heterogeneity in genetic composition may also account for differences in variability in thiocyanate levels within the same breed (Carlstrom 1965). Since the extent of enzyme activity is dependent on the concentration of the substrates present (thiocyanate in this case), it is not surprising that lactoperoxidase activity was highest in the Goudali breed and also higher during the dry than during the rainy season.

Seasonal changes such as temperature and humidity also affect the chemical composition of milk. During the dry season when water is scarce and humidity low, the moisture content of the milk may have been significantly reduced. Lower moisture content may partially account for the increase in thiocyanate content during the dry season when compared to samples of the rainy season. This may also explain why enzyme activity was higher during the dry season. A higher enzyme activity during the dry season could be provoked by the increased availability of thiocyanate as a substrate for the characteristic enzymatic reaction of the LPS.

To optimise the efficacy of the LPS in preserving milk, extra quantities of thiocyanate and peroxide must be added to the milk. If the natural thiocyanate level in the milk is high, it implies that less thiocyanate will be needed to activate the LPS and consequently, the cost of preservation will be reduced. Furthermore, the addition of lower doses will render the milk more wholesome for human consumption (IDF 1994).

Conclusions

The results of this study indicate that LP activity and thiocyanate concentration in milk vary significantly within and between the predominant breeds of cows in western Cameroon.
Milk from the Goudali breed has the highest thiocyanate concentration and as such lesser quantities of thiocyanate need to be added. Similarly, lesser quantities of exogenous thiocyanate will be needed in the dry season irrespective of the breed, to optimise the efficacy of the LPS towards milk preservation in this part of the country.

Acknowledgements

The author wishes to thank the Association of Commonwealth Universities for financial assistance; MM. Micame Nicephor, Tebug Thomas and Ndebi George for their help in sample collection.

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